Jul 11, 2025

Public workspacescMTR-seq

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvmb7dog3p/v1
scMTR-seq
  • Peter Rugg-Gunn1,
  • Yang Wang1
  • 1Babraham Institute
  • Human Developmental Biology Initiative (HDBI)
Richard J Acton
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvmb7dog3p/v1
Protocol Citation: Peter Rugg-Gunn, Yang Wang 2025. scMTR-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmb7dog3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 11, 2025
Last Modified: July 11, 2025
Protocol Integer ID: 222312
Keywords: single cell sequencing, single cell multiomics, scMTR-seq, histone modifications, transcription factors, CUT&Tag, CUT&RUN, ChIP-seq, transcriptomes in single cell, transcriptome in the same single cell, specific for each target histone modification, multiple histone modification, sequencing technology, target histone modification, other chromatin, scmtr, protocol for scmtr, applied scmtr, transcriptome, situ reverse transcription, histone modification, including pluripotent stem cell differentiation, pluripotent stem cell differentiation, single cell, same single cell, high cell recovery, nuclear mrna, mrna, indexed proteina, transcription
Funders Acknowledgements:
Wellcome
Grant ID: 215116/Z/18/Z
Wellcome
Grant ID: 225839/Z/22/Z
BBSRC
Grant ID: BBS/E/B/000C0522
BBSRC
Grant ID: BBS/E/B/000C0523
MRC
Grant ID: MR/T011769/1
MRC
Grant ID: MR/V02969X/1
Abstract
Here, we describe a protocol for scMTR-seq (single cell multi-targets and mRNA sequencing), which is a multi-omics sequencing technology that can simultaneously profile multiple histone modifications or other chromatin-bound proteins and transcriptome in the same single cells. scMTR-seq has high sensitivity, high cell recovery and is highly scalable in terms of starting material requirement. The protocol has the following steps: i) pre-assemble antibodies specific for each target histone modification or protein with indexed proteinA-Tn5-adapters; ii) perform in situ Tn5-mediated tagmentation with indexed complexes; iii) capture nuclear mRNA with a barcoded poly-T primer followed by in situ reverse transcription; iv) label single cells using split-pool combinatorial barcoding. We have applied scMTR-seq to profile histone modifications and transcriptomes in single cells from a broad range of heterogeneous samples including pluripotent stem cell differentiation and mouse pre-implantation embryos.
Attachments
Icon for scMTR-seq_primers.xlsx
scMTR-seq_primers.xl...
17KB
Protocol materials
ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932
ReagentRoche Complete Protease Inhibitor EDTA-Free tablets Merck MilliporeSigma (Sigma-Aldrich)Catalog #5056489001
ReagentDPBS (no Ca, no Mg)ThermofisherCatalog #14190144
ReagentBovine Albumin Fraction V (7.5% solution)Thermo FisherCatalog #15260037
ReagentTrypan Blue Solution, 0.4%Thermo FisherCatalog #15250061
Reagent16% Formaldehyde (w/v) Methanol-freeThermo Fisher ScientificCatalog #28906
ReagentConcanavalin-coated magnetic beads Bangs LaboratoriesCatalog #BP531
ReagentEDTA (0.5 M), pH 8.0, RNase-freeThermo FisherCatalog #AM9260G
ReagentSodium Chloride (5M)Invitrogen - Thermo FisherCatalog #AM9760G
Reagent1M MgCl2Invitrogen - Thermo FisherCatalog #AM9530G
ReagentAnti-Histone H3 (acetyl K27) antibody - ChIP GradeAbcamCatalog #ab4729
ReagentAnti-Histone H3 (mono methyl K4) antibody - ChIP GradeAbcamCatalog #ab8895
ReagentAnti-Histone H3 (tri methyl K36) antibody - ChIP GradeAbcamCatalog #ab9050
ReagentTri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAbCell Signaling TechnologyCatalog #9733
ReagentAnti-Histone H3 (tri methyl K4) antibody - ChIP GradeAbcamCatalog #ab8580
ReagentAnti-Histone H3 (tri methyl K9) antibody - ChIP GradeAbcamCatalog #ab8898
ReagentNormal Rabbit IgG Control R&D SystemsCatalog #AB-105-C
ReagentPEG 6000 (Poly(ethylene glycol))Bio Basic Inc.Catalog #PB0432.SIZE.500g
ReagentdNTP Set 100 mM SolutionsThermo Fisher ScientificCatalog #R0182
ReagentProtector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335399001
ReagentSUPERaseIN RNase InhibitorThermo Fisher ScientificCatalog #AM2696
ReagentMaxima H Minus Reverse TranscriptaseThermo Fisher ScientificCatalog ##EP0741
ReagentT4 DNA Ligase - 100,000 unitsNew England BiolabsCatalog #M0202L
ReagentProtector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001
Reagent10% Triton X-100Merck MilliporeSigma (Sigma-Aldrich)
ReagentSDS, 10% Solution, RNase-freeThermo FisherCatalog #AM9822
Reagent1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
ReagentProteinase KThermo Fisher ScientificCatalog #EO0491
ReagentDynabeads MyOne Streptavidin C1Invitrogen - Thermo FisherCatalog #65001
ReagentPMSFMerck MilliporeSigma (Sigma-Aldrich)Catalog #P7626
ReagentFicoll PM‐400Merck MilliporeSigma (Sigma-Aldrich)Catalog #F5415-50ML
ReagentHotStart ReadyMix (KAPA HiFi PCR kit)Kapa BiosystemsCatalog #KK2601
Reagent Ampure XP beads Beckman CoulterCatalog #A63881
ReagentNEBNext® High-Fidelity 2X PCR Master MixNew England BiolabsCatalog #M0541
ReagentGlycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #G5516
Reagent1 M Hydroxyethyl piperazineethanesulfonic acid pH 7.9 (HEPES (K ))Merck MilliporeSigma (Sigma-Aldrich)Catalog #H3375
Reagent1 M Potassium Chloride (KCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3911
Reagent2 M Spermidine Merck MilliporeSigma (Sigma-Aldrich)Catalog #S2501
ReagentPBS Thermo Fisher ScientificCatalog #28374
ReagentGlycine
Reagent1 M Hydroxyethyl piperazineethanesulfonic acid pH 7.5 (HEPES (Na ))Merck MilliporeSigma (Sigma-Aldrich)Catalog #H3375
Reagent5M NaClAmbionCatalog #AM9760G
Reagent1 M Calcium Chloride (CaCl2)Fisher ScientificCatalog #BP510
Reagent1 M Manganese Chloride (MnCl2)Merck MilliporeSigma (Sigma-Aldrich)Catalog #203734
ReagentTrizma® hydrochloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #T2319
ReagentNP-40 10% Merck MilliporeSigma (Sigma-Aldrich)Catalog #11332473001
Reagent10% Tween 20Bio-Rad LaboratoriesCatalog #1662404
ReagentTris HCl Buffer 1M Solution, Sterile pH 7.5Bio Basic Inc.Catalog #SD8124.SIZE.450ml
ReagentPierce™ Dimethylformamide (DMF), Sequencing gradeThermo Fisher ScientificCatalog #20672
Reagent5M NaCl solutionThermo Fisher ScientificCatalog #AM9759
ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
Troubleshooting
Solution Preparation

Overview of scMTR-seq

Solution Preparation


Protease Inhibitor Cocktail (PIC) (100x):

Dissolve 1 tablet of
ReagentRoche Complete Protease Inhibitor EDTA-Free tablets Merck MilliporeSigma (Sigma-Aldrich)Catalog #5056489001
in Amount500 µL ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932 , aliquot and store at Temperature-20 °C .

Avoid repeated freeze-thaw cycles
RNase Inhibitor (RI) mix (80x)

Mix:
  • ReagentSUPERaseIN RNase InhibitorThermo Fisher ScientificCatalog #AM2696
  • ReagentProtector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335399001
at a 1:1 ratio, and store at Temperature-20 °C
PBS with inhibitors (PBSI)

  • 1x PIC
  • 0.4x RI mix
  • 0.04% BSA ReagentBovine Albumin Fraction V (7.5% solution)Thermo FisherCatalog #15260037 diluted in ReagentDPBS (no Ca, no Mg)ThermofisherCatalog #14190144

eg: Amount2 µL PIC + Amount1 µL RI + Amount1.06 µL BSA +Amount196 µL DPBS 
2x Nuclei Extraction (NE) buffer
ABCD
ReagentsStockingWorking 1x 2x /μL
HEPES(K+) pH 7.91M 20 mM 1000
KCl 1M 10 mM 500
Spermidine 2M 0.5 mM 12.5
Triton-X100 10% (vol/vol) 0.1% 500
Glycerol 100% 20%10000
PIC*100x 2x500
RI*80x 2x625
Nuclease-free water 11862.5
  total 25000
* Only add 2x PIC and 2x RI mix just before use.
Prior to these additions, the buffer can be stored at Temperature4 °C for up to 3 months.
NB To achieve 40% glycerol in the 2x NE buffer, we typically add 12.5g of 100% glycerol.

  • Reagent1 M Hydroxyethyl piperazineethanesulfonic acid pH 7.9 (HEPES (K ))Merck MilliporeSigma (Sigma-Aldrich)Catalog #H3375
  • Reagent1 M Potassium Chloride (KCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3911
  • Reagent2 M Spermidine Merck MilliporeSigma (Sigma-Aldrich)Catalog #S2501
  • Reagent10% Triton X-100Merck MilliporeSigma (Sigma-Aldrich)
  • ReagentGlycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #G5516
  • PIC (100x)
  • RI (80x)
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932


5x Fixation quench buffer
ABCD
Reagents  Stocking Working 1x /μL
Glycine 2.5M 125 mM 50
Tris-HCl pH 8.0 1M 50 mM 50
Triton-X10010% 0.1% (in buffer) 2
BSA*7.5% 0.1% 13.4
PBS   84.6
  total 200
* Only add BSA just before use. Store Temperature4 °C .

  • ReagentGlycine
  • Reagent1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
  • Reagent10% Triton X-100Merck MilliporeSigma (Sigma-Aldrich)
  • ReagentBovine Albumin Fraction V (7.5% solution)Thermo FisherCatalog #15260037
  • ReagentPBS Thermo Fisher ScientificCatalog #28374


Wash Buffer
ABCD
Reagents  Stocking Working 1x /μL
HEPES(Na+) pH 7.5 1 M 20 mM 1000
NaCl5 M 150 mM 1500
Spermidine 2 M 0.5 mM 12.5
BSA*7.5% 1% 6666
PIC*100x 1x 500
RI*80x 1x 625
Nuclease-free water  39696
total50000
* Only add 1x PIC, 1x RI mix, and 1% BSA just before use.
Prior to these additions, buffer can be stored at Temperature4 °C for up to 3 months.


  • Reagent1 M Hydroxyethyl piperazineethanesulfonic acid pH 7.5 (HEPES (Na ))Merck MilliporeSigma (Sigma-Aldrich)Catalog #H3375
  • Reagent5M NaClAmbionCatalog #AM9760G
  • Reagent2 M Spermidine Merck MilliporeSigma (Sigma-Aldrich)Catalog #S2501
  • ReagentBovine Albumin Fraction V (7.5% solution)Thermo FisherCatalog #15260037
  • PIC (100x)
  • RI mix (80x)
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932

Binding Buffer
ABCD
Reagents  Stocking Working 1x /μL
HEPES (Na+) pH 7.51 M 20 mM 200
KCl 1 M 10 mM 100
CaCl2 1 M 1 mM 10
MnCl2 1 M 1 mM 10
Nuclease-free water 9680
  total 10000
Store the buffer at Temperature4 °C for up to 6 months

  • Reagent1 M Hydroxyethyl piperazineethanesulfonic acid pH 7.5 (HEPES (Na ))Merck MilliporeSigma (Sigma-Aldrich)Catalog #H3375
  • Reagent1 M Potassium Chloride (KCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3911
  • Reagent1 M Calcium Chloride (CaCl2)Fisher ScientificCatalog #BP510
  • Reagent1 M Manganese Chloride (MnCl2)Merck MilliporeSigma (Sigma-Aldrich)Catalog #203734
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932

Nuclei Isolation Buffer (NIB)
ABCDE
ReagentsStockingWorking 1x/μL
Trizma buffer pH 7.51 M 10 mM 500
NaCl5 M 10 mM 100
MgCl21 M 3 mM 150
NP-4010% 0.1% 500
Nuclease-free water  48750
  total 50000
Add PIC and RI mix just before use

  • ReagentTrizma® hydrochloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #T2319
  • ReagentSodium Chloride (5M)Invitrogen - Thermo FisherCatalog #AM9760G
  • Reagent1M MgCl2Invitrogen - Thermo FisherCatalog #AM9530G
  • ReagentNP-40 10% Merck MilliporeSigma (Sigma-Aldrich)Catalog #11332473001
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932

STE buffer
ABCD
Reagents  Stocking Working 1x /μL
Tris pH 8.0 1 M 10 mM 500
NaCl 5 M 50 mM 500
EDTA 0.5 M 1 mM 100
Nuclease-free water  48900
  total 50000
  • Reagent1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
  • ReagentSodium Chloride (5M)Invitrogen - Thermo FisherCatalog #AM9760G
  • ReagentEDTA (0.5 M), pH 8.0, RNase-freeThermo FisherCatalog #AM9260G
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932

1x B&W-T buffer
ABCD
Reagents  Stocking In buffer /μL
Tris pH 8.0 1M 5 mM 50
NaCl 5M 1 M 2000
EDTA 0.5 M 0.5 mM 10
Tween 20 10% 0.05% 50
Nuclease-free water  7890
  total 10000
  • Reagent1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
  • ReagentSodium Chloride (5M)Invitrogen - Thermo FisherCatalog #AM9760G
  • ReagentEDTA (0.5 M), pH 8.0, RNase-freeThermo FisherCatalog #AM9260G
  • Reagent10% Tween 20Bio-Rad LaboratoriesCatalog #1662404
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932


2x Binding & Washing buffer
ABCD
Reagents  Stocking In buffer /μL
Tris pH 8.0 1M 10 mM 10
NaCl 5M 2 M 400
EDTA 0.5 M 1 mM 2
Nuclease-free water  588
  total 1000
Add Superase RI before use (1μL RI to 45μL 2x Binding & Washing buffer)

  • Reagent1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
  • ReagentSodium Chloride (5M)Invitrogen - Thermo FisherCatalog #AM9760G
  • ReagentEDTA (0.5 M), pH 8.0, RNase-freeThermo FisherCatalog #AM9260G
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932

4x Tag Buffer for cDNA tagmentation
ABCD
ReagentsStockingIn Buffer /μL
Tris-HCl pH 7.51M40mM40
MgCl21M20mM20
Dimethylformamide (DMF)100%20%200
Nuclease-free water740
total1000

  • ReagentTris HCl Buffer 1M Solution, Sterile pH 7.5Bio Basic Inc.Catalog #SD8124.SIZE.450ml
  • Reagent1M MgCl2Invitrogen - Thermo FisherCatalog #AM9530G
  • ReagentPierce™ Dimethylformamide (DMF), Sequencing gradeThermo Fisher ScientificCatalog #20672
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932

Tn5 dilution buffer

ABCD
ReagentsStockingIn Buffer/uL
Tris-HCl pH7.51M10mM10
NaCL5M100mM20
Glycerol100%50% (0.625g)500
DTT1mM0.1M10
Nuclease-free water460
total1000

  • ReagentTris HCl Buffer 1M Solution, Sterile pH 7.5Bio Basic Inc.Catalog #SD8124.SIZE.450ml
  • Reagent5M NaCl solutionThermo Fisher ScientificCatalog #AM9759
  • ReagentGlycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #G5516
  • ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932

Prepare oligonucleotides for split pool barcoding
This section prepares the oligonucleotides that will add the sequence used to identify individual cells.

Example Annealed Barcodes & Linkers
Equipment:
  • PCR machine (thermocycler)
  • RNAse-free 96-well plates (Twin-Tec PRC plate, 96 Lobind, Semi-skirted)
Equipment
Eppendorf Twin tec PCR plates, 96 wells
NAME
96 well PCR plate
TYPE
Eppendorf
BRAND
EP0030128770
SKU
https://www.sigmaaldrich.com/GB/en/product/sigma/ep0030128770
LINK

Primers

Linker Primers

AB
NameSequence
Linker_1CGAATGCTCTGGCCTCTCAAGCACGTGGAT
Linker_2GGTCTGAGTTCGCACACGCTCCTCGAATCC
Linker_3AGTCGTACGCCGATGCGAAACATCGGCCAC


Barcode Primers

Split pool barcoding for cell identification, example primer sequence and index position:
BC1_#01
GTGCGAACTCAGACC CTGTAGCC ATCCACGTGCTTGAG
| Index |

BC1
AB
NameSequence
BC1_#01/5Phos/GTGCGAACTCAGACCAACGTGATATCCACGTGCTTGAG
BC1_#02/5Phos/GTGCGAACTCAGACCAAACATCGATCCACGTGCTTGAG
BC1_#03/5Phos/GTGCGAACTCAGACCACCACTGTATCCACGTGCTTGAG
BC1_#04/5Phos/GTGCGAACTCAGACCCATCAAGTATCCACGTGCTTGAG
BC1_#05/5Phos/GTGCGAACTCAGACCCTGTAGCCATCCACGTGCTTGAG
BC1_#06/5Phos/GTGCGAACTCAGACCAACCGAGAATCCACGTGCTTGAG
BC1_#07/5Phos/GTGCGAACTCAGACCAAGGTACAATCCACGTGCTTGAG
BC1_#08/5Phos/GTGCGAACTCAGACCACAGCAGAATCCACGTGCTTGAG
BC1_#09/5Phos/GTGCGAACTCAGACCACCTCCAAATCCACGTGCTTGAG
BC1_#10/5Phos/GTGCGAACTCAGACCACGCTCGAATCCACGTGCTTGAG
BC1_#11/5Phos/GTGCGAACTCAGACCACTATGCAATCCACGTGCTTGAG
BC1_#12/5Phos/GTGCGAACTCAGACCAGAGTCAAATCCACGTGCTTGAG
BC1_#13/5Phos/GTGCGAACTCAGACCAGATCGCAATCCACGTGCTTGAG
BC1_#14/5Phos/GTGCGAACTCAGACCCAATGGAAATCCACGTGCTTGAG
BC1_#15/5Phos/GTGCGAACTCAGACCCAGCGTTAATCCACGTGCTTGAG
BC1_#16/5Phos/GTGCGAACTCAGACCCATACCAAATCCACGTGCTTGAG
BC1_#17/5Phos/GTGCGAACTCAGACCCCAGTTCAATCCACGTGCTTGAG
BC1_#18/5Phos/GTGCGAACTCAGACCCGAACTTAATCCACGTGCTTGAG
BC1_#19/5Phos/GTGCGAACTCAGACCCGACTGGAATCCACGTGCTTGAG
BC1_#20/5Phos/GTGCGAACTCAGACCCTCAATGAATCCACGTGCTTGAG
BC1_#21/5Phos/GTGCGAACTCAGACCCTGAGCCAATCCACGTGCTTGAG
BC1_#22/5Phos/GTGCGAACTCAGACCGAATCTGAATCCACGTGCTTGAG
BC1_#23/5Phos/GTGCGAACTCAGACCGAGCTGAAATCCACGTGCTTGAG
BC1_#24/5Phos/GTGCGAACTCAGACCGATAGACAATCCACGTGCTTGAG
BC1_#25/5Phos/GTGCGAACTCAGACCGCTAACGAATCCACGTGCTTGAG
BC1_#26/5Phos/GTGCGAACTCAGACCGCTCGGTAATCCACGTGCTTGAG
BC1_#27/5Phos/GTGCGAACTCAGACCGGAGAACAATCCACGTGCTTGAG
BC1_#28/5Phos/GTGCGAACTCAGACCGGTGCGAAATCCACGTGCTTGAG
BC1_#29/5Phos/GTGCGAACTCAGACCGTACGCAAATCCACGTGCTTGAG
BC1_#30/5Phos/GTGCGAACTCAGACCGTCGTAGAATCCACGTGCTTGAG
BC1_#31/5Phos/GTGCGAACTCAGACCGTGTTCTAATCCACGTGCTTGAG
BC1_#32/5Phos/GTGCGAACTCAGACCTCTTCACAATCCACGTGCTTGAG
BC1_#33/5Phos/GTGCGAACTCAGACCTGGAACAAATCCACGTGCTTGAG
BC1_#34/5Phos/GTGCGAACTCAGACCTGGCTTCAATCCACGTGCTTGAG
BC1_#35/5Phos/GTGCGAACTCAGACCTGGTGGTAATCCACGTGCTTGAG
BC1_#36/5Phos/GTGCGAACTCAGACCTTCACGCAATCCACGTGCTTGAG
BC1_#37/5Phos/GTGCGAACTCAGACCAACTCACCATCCACGTGCTTGAG
BC1_#38/5Phos/GTGCGAACTCAGACCAAGAGATCATCCACGTGCTTGAG
BC1_#39/5Phos/GTGCGAACTCAGACCAAGGACACATCCACGTGCTTGAG
BC1_#40/5Phos/GTGCGAACTCAGACCAATCCGTCATCCACGTGCTTGAG
BC1_#41/5Phos/GTGCGAACTCAGACCACACGACCATCCACGTGCTTGAG
BC1_#42/5Phos/GTGCGAACTCAGACCAGCCATGCATCCACGTGCTTGAG
BC1_#43/5Phos/GTGCGAACTCAGACCATCATTCCATCCACGTGCTTGAG
BC1_#44/5Phos/GTGCGAACTCAGACCCACCTTACATCCACGTGCTTGAG
BC1_#45/5Phos/GTGCGAACTCAGACCCCTAATCCATCCACGTGCTTGAG
BC1_#46/5Phos/GTGCGAACTCAGACCCCTCTATCATCCACGTGCTTGAG
BC1_#47/5Phos/GTGCGAACTCAGACCGAACAGGCATCCACGTGCTTGAG
BC1_#48/5Phos/GTGCGAACTCAGACCGATGAATCATCCACGTGCTTGAG

BC2
AB
NameSequence
BC2_#01/5Phos/CATCGGCGTACGACTAACGTGATGGATTCGAGGAGCGT
BC2_#02/5Phos/CATCGGCGTACGACTAAACATCGGGATTCGAGGAGCGT
BC2_#03/5Phos/CATCGGCGTACGACTACCACTGTGGATTCGAGGAGCGT
BC2_#04/5Phos/CATCGGCGTACGACTCATCAAGTGGATTCGAGGAGCGT
BC2_#05/5Phos/CATCGGCGTACGACTCTGTAGCCGGATTCGAGGAGCGT
BC2_#06/5Phos/CATCGGCGTACGACTAACCGAGAGGATTCGAGGAGCGT
BC2_#07/5Phos/CATCGGCGTACGACTAAGGTACAGGATTCGAGGAGCGT
BC2_#08/5Phos/CATCGGCGTACGACTACAGCAGAGGATTCGAGGAGCGT
BC2_#09/5Phos/CATCGGCGTACGACTACCTCCAAGGATTCGAGGAGCGT
BC2_#10/5Phos/CATCGGCGTACGACTACGCTCGAGGATTCGAGGAGCGT
BC2_#11/5Phos/CATCGGCGTACGACTACTATGCAGGATTCGAGGAGCGT
BC2_#12/5Phos/CATCGGCGTACGACTAGAGTCAAGGATTCGAGGAGCGT
BC2_#13/5Phos/CATCGGCGTACGACTAGATCGCAGGATTCGAGGAGCGT
BC2_#14/5Phos/CATCGGCGTACGACTCAATGGAAGGATTCGAGGAGCGT
BC2_#15/5Phos/CATCGGCGTACGACTCAGCGTTAGGATTCGAGGAGCGT
BC2_#16/5Phos/CATCGGCGTACGACTCATACCAAGGATTCGAGGAGCGT
BC2_#17/5Phos/CATCGGCGTACGACTCCAGTTCAGGATTCGAGGAGCGT
BC2_#18/5Phos/CATCGGCGTACGACTCGAACTTAGGATTCGAGGAGCGT
BC2_#19/5Phos/CATCGGCGTACGACTCGACTGGAGGATTCGAGGAGCGT
BC2_#20/5Phos/CATCGGCGTACGACTCTCAATGAGGATTCGAGGAGCGT
BC2_#21/5Phos/CATCGGCGTACGACTCTGAGCCAGGATTCGAGGAGCGT
BC2_#22/5Phos/CATCGGCGTACGACTGAATCTGAGGATTCGAGGAGCGT
BC2_#23/5Phos/CATCGGCGTACGACTGAGCTGAAGGATTCGAGGAGCGT
BC2_#24/5Phos/CATCGGCGTACGACTGATAGACAGGATTCGAGGAGCGT
BC2_#25/5Phos/CATCGGCGTACGACTGCTAACGAGGATTCGAGGAGCGT
BC2_#26/5Phos/CATCGGCGTACGACTGCTCGGTAGGATTCGAGGAGCGT
BC2_#27/5Phos/CATCGGCGTACGACTGGAGAACAGGATTCGAGGAGCGT
BC2_#28/5Phos/CATCGGCGTACGACTGGTGCGAAGGATTCGAGGAGCGT
BC2_#29/5Phos/CATCGGCGTACGACTGTACGCAAGGATTCGAGGAGCGT
BC2_#30/5Phos/CATCGGCGTACGACTGTCGTAGAGGATTCGAGGAGCGT
BC2_#31/5Phos/CATCGGCGTACGACTGTGTTCTAGGATTCGAGGAGCGT
BC2_#32/5Phos/CATCGGCGTACGACTTCTTCACAGGATTCGAGGAGCGT
BC2_#33/5Phos/CATCGGCGTACGACTTGGAACAAGGATTCGAGGAGCGT
BC2_#34/5Phos/CATCGGCGTACGACTTGGCTTCAGGATTCGAGGAGCGT
BC2_#35/5Phos/CATCGGCGTACGACTTGGTGGTAGGATTCGAGGAGCGT
BC2_#36/5Phos/CATCGGCGTACGACTTTCACGCAGGATTCGAGGAGCGT
BC2_#37/5Phos/CATCGGCGTACGACTAACTCACCGGATTCGAGGAGCGT
BC2_#38/5Phos/CATCGGCGTACGACTAAGAGATCGGATTCGAGGAGCGT
BC2_#39/5Phos/CATCGGCGTACGACTAAGGACACGGATTCGAGGAGCGT
BC2_#40/5Phos/CATCGGCGTACGACTAATCCGTCGGATTCGAGGAGCGT
BC2_#41/5Phos/CATCGGCGTACGACTACACGACCGGATTCGAGGAGCGT
BC2_#42/5Phos/CATCGGCGTACGACTAGCCATGCGGATTCGAGGAGCGT
BC2_#43/5Phos/CATCGGCGTACGACTATCATTCCGGATTCGAGGAGCGT
BC2_#44/5Phos/CATCGGCGTACGACTCACCTTACGGATTCGAGGAGCGT
BC2_#45/5Phos/CATCGGCGTACGACTCCTAATCCGGATTCGAGGAGCGT
BC2_#46/5Phos/CATCGGCGTACGACTCCTCTATCGGATTCGAGGAGCGT
BC2_#47/5Phos/CATCGGCGTACGACTGAACAGGCGGATTCGAGGAGCGT
BC2_#48/5Phos/CATCGGCGTACGACTGATGAATCGGATTCGAGGAGCGT

BC3
AB
NameSequence
BC3_#01CAGACGTGTGCTCTTCCGATCTAACGTGATGTGGCCGATGTTTCG
BC3_#02CAGACGTGTGCTCTTCCGATCTAAACATCGGTGGCCGATGTTTCG
BC3_#03CAGACGTGTGCTCTTCCGATCTACCACTGTGTGGCCGATGTTTCG
BC3_#04CAGACGTGTGCTCTTCCGATCTCATCAAGTGTGGCCGATGTTTCG
BC3_#05CAGACGTGTGCTCTTCCGATCTCTGTAGCCGTGGCCGATGTTTCG
BC3_#06CAGACGTGTGCTCTTCCGATCTAACCGAGAGTGGCCGATGTTTCG
BC3_#07CAGACGTGTGCTCTTCCGATCTAAGGTACAGTGGCCGATGTTTCG
BC3_#08CAGACGTGTGCTCTTCCGATCTACAGCAGAGTGGCCGATGTTTCG
BC3_#09CAGACGTGTGCTCTTCCGATCTACCTCCAAGTGGCCGATGTTTCG
BC3_#10CAGACGTGTGCTCTTCCGATCTACGCTCGAGTGGCCGATGTTTCG
BC3_#11CAGACGTGTGCTCTTCCGATCTACTATGCAGTGGCCGATGTTTCG
BC3_#12CAGACGTGTGCTCTTCCGATCTAGAGTCAAGTGGCCGATGTTTCG
BC3_#13CAGACGTGTGCTCTTCCGATCTAGATCGCAGTGGCCGATGTTTCG
BC3_#14CAGACGTGTGCTCTTCCGATCTCAATGGAAGTGGCCGATGTTTCG
BC3_#15CAGACGTGTGCTCTTCCGATCTCAGCGTTAGTGGCCGATGTTTCG
BC3_#16CAGACGTGTGCTCTTCCGATCTCATACCAAGTGGCCGATGTTTCG
BC3_#17CAGACGTGTGCTCTTCCGATCTCCAGTTCAGTGGCCGATGTTTCG
BC3_#18CAGACGTGTGCTCTTCCGATCTCGAACTTAGTGGCCGATGTTTCG
BC3_#19CAGACGTGTGCTCTTCCGATCTCGACTGGAGTGGCCGATGTTTCG
BC3_#20CAGACGTGTGCTCTTCCGATCTCTCAATGAGTGGCCGATGTTTCG
BC3_#21CAGACGTGTGCTCTTCCGATCTCTGAGCCAGTGGCCGATGTTTCG
BC3_#22CAGACGTGTGCTCTTCCGATCTGAATCTGAGTGGCCGATGTTTCG
BC3_#23CAGACGTGTGCTCTTCCGATCTGAGCTGAAGTGGCCGATGTTTCG
BC3_#24CAGACGTGTGCTCTTCCGATCTGATAGACAGTGGCCGATGTTTCG
BC3_#25CAGACGTGTGCTCTTCCGATCTGCTAACGAGTGGCCGATGTTTCG
BC3_#26CAGACGTGTGCTCTTCCGATCTGCTCGGTAGTGGCCGATGTTTCG
BC3_#27CAGACGTGTGCTCTTCCGATCTGGAGAACAGTGGCCGATGTTTCG
BC3_#28CAGACGTGTGCTCTTCCGATCTGGTGCGAAGTGGCCGATGTTTCG
BC3_#29CAGACGTGTGCTCTTCCGATCTGTACGCAAGTGGCCGATGTTTCG
BC3_#30CAGACGTGTGCTCTTCCGATCTGTCGTAGAGTGGCCGATGTTTCG
BC3_#31CAGACGTGTGCTCTTCCGATCTGTGTTCTAGTGGCCGATGTTTCG
BC3_#32CAGACGTGTGCTCTTCCGATCTTCTTCACAGTGGCCGATGTTTCG
BC3_#33CAGACGTGTGCTCTTCCGATCTTGGAACAAGTGGCCGATGTTTCG
BC3_#34CAGACGTGTGCTCTTCCGATCTTGGCTTCAGTGGCCGATGTTTCG
BC3_#35CAGACGTGTGCTCTTCCGATCTTGGTGGTAGTGGCCGATGTTTCG
BC3_#36CAGACGTGTGCTCTTCCGATCTTTCACGCAGTGGCCGATGTTTCG
BC3_#37CAGACGTGTGCTCTTCCGATCTAACTCACCGTGGCCGATGTTTCG
BC3_#38CAGACGTGTGCTCTTCCGATCTAAGAGATCGTGGCCGATGTTTCG
BC3_#39CAGACGTGTGCTCTTCCGATCTAAGGACACGTGGCCGATGTTTCG
BC3_#40CAGACGTGTGCTCTTCCGATCTAATCCGTCGTGGCCGATGTTTCG
BC3_#41CAGACGTGTGCTCTTCCGATCTACACGACCGTGGCCGATGTTTCG
BC3_#42CAGACGTGTGCTCTTCCGATCTAGCCATGCGTGGCCGATGTTTCG
BC3_#43CAGACGTGTGCTCTTCCGATCTATCATTCCGTGGCCGATGTTTCG
BC3_#44CAGACGTGTGCTCTTCCGATCTCACCTTACGTGGCCGATGTTTCG
BC3_#45CAGACGTGTGCTCTTCCGATCTCCTAATCCGTGGCCGATGTTTCG
BC3_#46CAGACGTGTGCTCTTCCGATCTCCTCTATCGTGGCCGATGTTTCG
BC3_#47CAGACGTGTGCTCTTCCGATCTGAACAGGCGTGGCCGATGTTTCG
BC3_#48CAGACGTGTGCTCTTCCGATCTGATGAATCGTGGCCGATGTTTCG


The following sub-steps describe annealing linker primers with barcode primers in RNAse-free 96-well plates (Twin-Tec PRC plate, 96 Lobind, Semi-skirted) to a total volume of Amount10 µL per well. At the end of Go to the resulting plate will contain oligos with the following concentrations:

BC1-Linker_1: in round 1 plates, containing:
- Amount9 µL Linker_1 and
- Amount10 µL BC1 barcodes
BC2-Linker_2: in round 2 plates, containing:
- Amount11 µL Linker_2 and
- Amount12 µL BC2 barcodes
BC3-Linker_3: in round 3 plates, containing:
- Amount13 µL Linker_3 and
- Amount14 µL BC3 barcodes

To prepare stock of 9 plates of 48 barcodes: 

For each linker-barcode, prepare Amount10 µL /well x 9 x 1.1 = Amount100 µL ; 

  • BC1-Linker1:
- Linker_1 (Concentration1 millimolar (mM) stock) Amount0.9 µL
- BC1 (Concentration100 micromolar (µM) stock) Amount10 µL
- STE buffer Amount89.1 µL (Go to STE )
  • BC2-Linker2:
- Linker_2 (Concentration1 millimolar (mM) stock) Amount1.1 µL
- BC2 (Concentration100 micromolar (µM) stock) Amount12 µL
- STE buffer Amount86.9 µL
  • BC3-Linker3:
- Linker_3 (Concentration1 millimolar (mM) stock) Amount1.3 µL
- BC3 (Concentration100 micromolar (µM) stock) Amount14 µL
- STE buffer Amount84.7 µL

Prepare linker stocks (x 48 x 1.1 = x 53; in each round, linker is the same one) 

  • Linker_1 Amount47.7 µL + STE buffer Amount4722.3 µL
  • Linker_2 Amount58.3 µL + STE buffer Amount4605.7 µL
  • Linker_3 Amount68.9 µL + STE buffer Amount4489.1 µL
Aliquot diluted linker into 48 wells: Linker_1 Amount90 µL ; Linker_2 Amount88 µL ; Linker_3 Amount86 µL . 

  • Add barcodes of BC1 (1-48) Amount10 µL to Linker_1
  • Add barcodes of BC2 (1-48) Amount12 µL to Linker_2
  • Add barcodes of BC3 (1-48) Amount14 µL to Linker_3
Place sealed plates with annealing linker and barcodes in PCR machine with the programme:

Temperature95 °C Duration00:02:00 , cooling down to Temperature20 °C at Temperature-1 °C /min

Aliquot mixture Amount10 µL to each well into 96-well plates
Seal plates and store at Temperature-20 °C .
Solution Preparation
Equipment:
  • PCR machine (thermocycler)

Primers
Barcoding for antibody Identification, with example primer sequence and index position:


AB
NameSequence
MEB_dU_#01/5Phos/AGGCCAGAGCATTCGAATGGNNNNNNN/ideoxyU/AGATGTGTATAAGAGACAG
MEB_dU_#02/5Phos/AGGCCAGAGCATTCGTGAGANNNNNNN/ideoxyU/AGATGTGTATAAGAGACAG
MEB_dU_#03/5Phos/AGGCCAGAGCATTCGCCATANNNNNNN/ideoxyU/AGATGTGTATAAGAGACAG
MEB_dU_#04/5Phos/AGGCCAGAGCATTCGGGTGTNNNNNNN/ideoxyU/AGATGTGTATAAGAGACAG
ME_R/5Phos/C*T*G*T*C*T*C*T*T*A*T*A*C*A*/3ddC/
ME_ATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
*N indicates a phosphorothioate bond (primer sequences are in the IDT DNA format).

Dilute oligonucleotides to Concentration200 micromolar (µM) in STE buffer (Go to STE )
pA-Tn5-MeBdU-MeR
For each of the four MEB_dU oligonucleotides, in separate tubes mix:
  • Amount2 µL Concentration200 micromolar (µM) phosphorylated Mosaic end adapter B dU (MEB_dU_#N)
  • Amount2 µL Concentration200 micromolar (µM) blocked Mosaic end-reverse (ME_R) oligonucleotides
to generate Concentration100 micromolar (µM) pre-annealed MEBdU-MER
pA-Tn5-MeA-MeR
Mix:
  • Amount2 µL Concentration200 micromolar (µM) Mosaic end adapter A (ME_A)
  • Amount2 µL Concentration200 micromolar (µM) blocked Mosaic end-reverse (ME-R) oligonucleotides
to generate Concentration100 micromolar (µM) pre-annealed MEA-MER
Place the tubes in a thermocycler and run Temperature95 °C Duration00:02:00 , slowly cool to Temperature20 °C with ramp rate of Temperature-1 °C /min

2m
Mix Amount4 µL Concentration100 micromolar (µM) pre-annealed MEBdU-MER or MEA-MER with Amount40 µL of Concentration5.5 micromolar (µM) pA-Tn5 fusion protein and incubate on a rotating platform for Duration01:00:00 at room temperature. Store at Temperature-20 °C for up to 1 year.


Note
We use home-made pA-Tn5 fusion protein generated from plasmid #124601 from Addgene https://www.addgene.org/124601/ following this protocol:
Citation
Terri Bryson, Steven Henikoff. 3XFlag-pATn5 Protein Purification and MEDS-loading (5x scale, 2L volume). protocols.io.
https://protocols.io/view/3xflag-patn5-protein-purification-and-meds-loading-8yrhxv6
LINK
Commercially available alternatives are:
  • ReagentpA-Tn5 Transposase (10 µg)Active MotifCatalog #53161
  • Reagent CUTANA™ Uncharged pAG-Tn5 for CUT&TagEpiCypherCatalog #15-1025

1h
Single-cell dissociation
Equipment:
  • Centrifuge with swing-bucket rotor
  • Amount1.5 mL protein lobind tube

Dissociate samples into single-cell suspension, and transfer cells to Amount1.5 mL protein lobind tube
Wash cells twice with PBSI (Go to PBSI ) by centrifuging cells in a swing-bucket rotor at Centrifigation300 x g, 00:03:00 , and remove the supernatant.

Nuclei Preparation
To prepare nuclei:

Measure the the volume of the the single cell suspension and add PBSI (Go to PBSI ) to cells to a final volume of Amount80 µL

Add Amount80 µL 2x NE buffer (Go to 2x NE Nuffer ) (with 2x PIC and 2x RI)
briefly vortex to mix and put cells on ice for Duration00:10:00 (adjust incubation time if needed; ReagentTrypan Blue Solution, 0.4%Thermo FisherCatalog #15250061 , trypan blue positive > 95%). 
 
Light Fixation and Quench
To help maintain intact nuclei for split-pool barcoding, we use a light fixation with 0.2% formaldehyde.

Equipment:
  • Centrifuge with swing bucket rotor
  • (optional) Mr. Frosty container

Fixation wash buffer

ABC
ReagentStocking/µL
Base Wash Buffer867
BSA7.5%133
PIC100x10
RI80x12.5


Quickly spin (Centrifigation100 x g, 00:00:05 ) to collect solution into the bottom, add Amount2 µL Reagent16% Formaldehyde (w/v) Methanol-freeThermo Fisher ScientificCatalog #28906 to final 0.2%, immediately tap to mix and start stopwatch. Incubate for Duration00:05:00 exactly .

Quickly spin (Centrifigation100 x g, 00:00:05 ) to collect solution into the bottom, add Amount40 µL 5x Fixation Quench Buffer (Go to 5x Fixation Quench Buffer ) to quench nuclei, invert to mix, place on ice for Duration00:05:00 - Duration00:10:00 .

Centrifuge nuclei in a swing-bucket rotor (pre-cooled) at Centrifigation600 x g, 4°C, 00:03:00 , carefully take out tubes and remove Amount180 µL from the top.
Leave ~Amount20 µL in the tube without disturbing nuclei at the bottom.
Add Amount100 µL Fixation wash buffer to re-suspend cells, invert to mix.

Note
Optional Stopping Point

Quickly spin Centrifigation100 x g, 00:00:05 , add Amount15 µL 100% DMSO to each sample (10-15% in final), invert to mix. Quickly spin Centrifigation100 x g, 00:00:05 , place samples in a Mr. Frosty container and store at Temperature-80 °C overnight to freeze samples. The next day, samples are ready for shipment on dry ice or can be stored at Temperature-80 °C freezer for a longer term. 


Pause
Binding nuclei to beads
15m
To immobilise nuclei, we bind the lightly-fixed nuclei with Concanavalin A (ConA)-coated magnetic beads.

Equipment:
  • Magnet stand

Beads Preparation
Transfer (5 x N* x 1.1) μL
ReagentConcanavalin-coated magnetic beads Bangs LaboratoriesCatalog #BP531 bead slurry into a Amount1.5 mL tube , wash twice with Amount1 mL binding buffer, mix by pipetting, and place the tube on a magnetic stand to allow the mixture to clear, and drain liquid completely.

*where N is the number of samples

Note
Using more bead slurry could alleviate the formation of clumps. We typically use around Amount1 µL per 1,000 cells.


Re-suspend beads in (5 x N x 1.1) μL binding buffer, and hold on ice until nuclei are ready.

Preparing

Thawing Wash Buffer:

ABCD
ReagentsStockingWorking/μL
Base Wash Buffer867
BSA7.5%1%133
PIC100x1x10
RI80x0.4x5


  • Amount867 µL wash buffer (Go to wash buffer )
  • Amount133 µL ReagentBovine Albumin Fraction V (7.5% solution)Thermo FisherCatalog #15260037
  • Amount5 µL RI mix
  • Amount10 µL PIC 

Thawing Wash buffer + EDTA:

ABCD
ReagentsStockingWorking/μL
Thawing Wash Buffer500
EDTA0.5 M2 µM2


  • Amount500 µL Thawing Wash Buffer
  • Amount2 µL ReagentEDTA (0.5 M), pH 8.0, RNase-freeThermo FisherCatalog #AM9260G

Thaw nuclei on ice, add Amount500 µL Thawing Wash Buffer
Slowly add prepared ConA beads from step 12.2 into cell suspension while vortexing gently Shaker1100 rpm Vortex Gently , place tubes on an end-over-end rotator for Duration00:05:00 - Duration00:10:00 TemperatureRoom temperature

15m
Place the tubes on a magnetic stand to allow the mixtures to clear. Carefully discard the liquid (taking care due to surface tension).
Wash once with Amount100 µL Thawing Wash Buffer + EDTA while tubes are on the magnetic stand.
DNA tagmentation
The steps below describe how to simultaneously tagment and profile multiple indexed antibodies using scMTR-seq. We use indexed MEBdU to multiplex each primary antibody of choice with different pA-Tn5-MEBdU-MER. To minimise cross-contamination between antibodies, we then block any unreacted pA-Tn5-MEBdu_MER by incubation with normal IgG. We have successfully profiled five different histone modifications simaltaenously with only minor off-target signals detected. Including an additional IgG profile helps to identify and further reduce off-target signals using computational methods.

As an alternative approach, we have also performed tagmentation in multiple sequential steps, rather than simultaneously (see step 13.12). Sequential profiling increases hands-on time and may lead to greater loss of nuclei, although the advantages of this alternative approach include reduced off-target signals and the opportunity to profile more targets in different rounds.
Solution Preparation


Equipment:
  • end-over-end rotator
  • cold room
  • PCR machine (thermocycler)

Preparing
High RI wash 300 buffer

ABC
ReagentsStocking/μL
Base Wash Buffer867
NaCl5M30
BSA7.5%133
PIC100x10
RI80x12.5


  • Amount867 µL wash buffer (Go to wash buffer )
  • Amount30 µL Concentration5 Molarity (M) ReagentSodium Chloride (5M)Invitrogen - Thermo FisherCatalog #AM9760G
  • Amount133 µL ReagentBovine Albumin Fraction V (7.5% solution)Thermo FisherCatalog #15260037
  • Amount12.5 µL RI mix
  • Amount10 µL PIC

(make Amount100 µL per sample) 
Low RI Wash 300 buffer

ABC
ReagentsStocking/μL
Base Wash Buffer867
NaCl5 M30
BSA7.5%133
PIC100x10
RI80x5


  • Amount867 µL wash buffer (Go to wash buffer )
  • Amount30 µL Concentration5 Molarity (M) ReagentSodium Chloride (5M)Invitrogen - Thermo FisherCatalog #AM9760G
  • Amount133 µL ReagentBovine Albumin Fraction V (7.5% solution)Thermo FisherCatalog #15260037
  • Amount10 µL PIC
  • Amount5 µL RI mix

(make Amount100 µL per sample) 
Tag buffer

ABC
ReagentStocking/μL
Base Wash Buffer867
NaCl5 M30
BSA7.5%13.3
PIC100x10
RI80x25
Nuclease-free water119.7
MgCl21M10

  • Amount867 µL wash buffer (Go to wash buffer )
  • Amount30 µL Concentration5 Molarity (M) ReagentSodium Chloride (5M)Invitrogen - Thermo FisherCatalog #AM9760G
  • Amount13.3 µL ReagentBovine Albumin Fraction V (7.5% solution)Thermo FisherCatalog #15260037
  • Amount25 µL RI mix
  • Amount10 µL PIC
  • Amount119.7 µL ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932
  • Amount10 µL Concentration1 Molarity (M) Reagent1M MgCl2Invitrogen - Thermo FisherCatalog #AM9530G

(make Amount100 µL per sample) 

Pre-assemble selected primary antibody (Ab) and indexed pA-Tn5-MeBdU-MeR (0.5-1h). We list below several antibodies that we have used successfully in scMTR-seq. Alternative antibodies can be tested first using bulk CUT&Tag to check for signal specificity.

Example Antibodies:
  • ReagentAnti-Histone H3 (tri methyl K4) antibody - ChIP GradeAbcamCatalog #ab8580
  • ReagentAnti-Histone H3 (tri methyl K9) antibody - ChIP GradeAbcamCatalog #ab8898
  • ReagentAnti-Histone H3 (acetyl K27) antibody - ChIP GradeAbcamCatalog #ab4729
  • ReagentAnti-Histone H3 (mono methyl K4) antibody - ChIP GradeAbcamCatalog #ab8895
  • ReagentTri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAbCell Signaling TechnologyCatalog #9733
  • ReagentAnti-Histone H3 (tri methyl K36) antibody - ChIP GradeAbcamCatalog #ab9050

Mix:
  • Amount0.5 µL Concentration1 µg/µL antibody
  • Amount0.5 µL pA-Tn5-MeBdU-MeR
  • Amount4.25 µL High RI wash 300 buffer

place tubes on an end-over-end rotator
Centrifigation10 rpm, Room temperature, 01:00:00 , end-over-end rotator


1h
Block any remaining unreacted proteinA-Tn5:

Add Amount1 µL Concentration1 µg/µL ReagentNormal Rabbit IgG Control R&D SystemsCatalog #AB-105-C and incubate TemperatureRoom temperature for Duration00:30:00

30m
Mix pre-assembled Ab-pA-Tn5-MeBdU-MeR (Amount6 µL x n of indexed antibodies) with ConA-bound nuclei, add High RI wash 300 to Amount99.6 µL , and then add Amount0.4 µL Concentration0.5 Molarity (M) EDTA.

Gently mix, briefly spin and incubate in cold room (Temperature4 °C ) DurationOvernight at 25-30 rpm on rotator or 1-2 hrs at TemperatureRoom temperature

2h
Overnight
Wash three times with Low RI Wash 300 buffer, using the magnetic stand, and invert to mix between washes
Re-suspend with Amount50 µL Tag buffer, gently mix and place in PCR machine, incubate Temperature37 °C for Duration01:00:00  

1h
Directly place tube on pre-cooled rack, and add Amount1.67 µL Concentration0.5 Molarity (M) EDTA each sample to stop tagmentation, gently tap to mix 
Add Amount8 µL ReagentBovine Albumin Fraction V (7.5% solution)Thermo FisherCatalog #15260037 to each sample to avoid clumps forming. Mix by pipetting or gently tapping, place on ice for Duration00:05:00

5m
(Optional)
For sequential tagmentation, wash twice with Low RI wash 300 buffer after adding stop buffer, and repeat from Go to to step 13.11.

Optional
In situ Reverse Transcription
17m 12s
In situ reverse transcribe polyA mRNA with biotinylated dT RT primers in the nuclei.

Equipment:
  • PCR machine

Primers


AB
NameSequence
RT_#01/5Phos/AGGCCAGAGCATTCGAGCAANNNNNNNN /iBiodT/TTTTTTTTTTTTTTVN
RT_#02/5Phos/AGGCCAGAGCATTCGCCTAANNNNNNNN /iBiodT/TTTTTTTTTTTTTTVN
RT_#03/5Phos/AGGCCAGAGCATTCGGTCGANNNNNNNN /iBiodT/TTTTTTTTTTTTTTVN
RT_#04/5Phos/AGGCCAGAGCATTCGTGTGANNNNNNNN /iBiodT/TTTTTTTTTTTTTTVN

Preparing
NIB-RI buffer

  • Amount867 µL NIB buffer (Go to Nuclei Isolation Buffer )
  • Amount133 µL ReagentBovine Albumin Fraction V (7.5% solution)Thermo FisherCatalog #15260037
  • Amount5 µL RI mix
  • Amount10 µL PIC

(make Amount350 µL per sample)

RT mix:
ABCD
ReagentStockingWorking 1x1x volume /μL
RT buffer5x1x10
PEG 600050 %15 %15
RT_#N primer100 μM10 μM5
dNTPs25 mM/each0.5 mM1
RNAse Protector40 U/μL0.5 U/μL0.625
Superase RI20 U/μL0.5 U/μL1.25
Maxima H Minus RT200 U/μL20 U/μl5
Nuclease-free water 12.125
total 50

  • ReagentPEG 6000 (Poly(ethylene glycol))Bio Basic Inc.Catalog #PB0432.SIZE.500g
  • RT_#N
  • ReagentdNTP Set 100 mM SolutionsThermo Fisher ScientificCatalog #R0182
  • ReagentProtector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335399001
  • ReagentSUPERaseIN RNase InhibitorThermo Fisher ScientificCatalog #AM2696
  • ReagentMaxima H Minus Reverse TranscriptaseThermo Fisher ScientificCatalog ##EP0741
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932

Wash in situ-tagged nuclei twice with Amount100 µL NIB-RI buffer. 
Add Amount10 µL of RT mix per 10,000 nuclei to each tube, gently mix and briefly centrifuge

  • Run following programme to synthesise the first strand of cDNA:  Temperature50 °C Duration00:10:00
  • 3 cycles of annealing:
Temperature8 °C Duration00:00:12
Temperature15 °C Duration00:00:45
Temperature20 °C Duration00:00:45
Temperature30 °C Duration00:00:30
Temperature42 °C Duration00:02:00
Temperature50 °C Duration00:03:00
End cycles
  • Temperature50 °C 5 min
  • Temperature4 °C Hold
17m 12s
Add Amount50 µL NIB-RI to dilute sample, place tubes on PCR magnet and remove liquid 
Wash once with Amount100 µL NIB-RI and remove liquid
Split-pool barcoding
3h 14m
Three rounds of split-pool barcoding were used for barcoding single cells. As there is 48 different barcodes for each round in our assay, this will generate over 110,000 barcodes combinations (# of BC1 x # of BC2 x # of BC3) after three rounds of the split-pool barcoding procedure. Both cDNA and tagged gDNA molecules will get the same combination of barcodes within the same single nuclei.

Note
To ensure there are enough barcode combinations for barcoding each single cell/nuclei with a unique cell barcode, we adjust the conditions to keep the collision rate below 5%. The collision rate is calculated by dividing the number of cells or nuclei by the number of barcode combinations.

The number of cells or nuclei: as the recovery rate from the starting point is normally ~20-40%, we use the number of starting cells multiplied by the recovery rate to estimate the number of cells/nuclei.

The number of barcode combinations: in addition to the split-pool steps, we could use different RT primers and MEB adapters for different samples, and different nuclei could be aliquoted into different sub-libraries and amplified with different i5-i7 primer sets, so all these steps will contribute to the total number of barcodes combinations. Therefore, the number of barcode combinations = the number of RT primers or MEB adapters x number of BC1 x number of BC2 x number of BC3 x number of sub-libraries.

In practice, we normally adjust the number of RT primers and MEB adapters used and the number of sub-libraries to keep the collision rate below 5%.

Example calculations are included in the attached spreadsheet.






Equipment:
  • Plate centrifuge
  • Multichannel pipettor
  • Thermomixer

Primers

AB
NameSequence
Blocker_1ATCCACGTGCTTGAGAGGCCAGAGCATTCG
Blocker_2GGATTCGAGGAGCGTGTGCGAACTCAGACC
Blocker_3GTGGCCGATGTTTCGCATCGGCGTACGACT


Preparing
Hybridisation mix: (Amount2 mL per sample)
ABCD
ReagentsStockingWorking 1x/μl
T4 ligation buffer10x1x200
RNase Inhibitor40 U/μL0.32 U/μl16
SUPERase RI20 U/μL0.05 U/μl5
PIC100x1x20
Triton X-10010% (vol/vol)0.1%20
BSA7.5%0.1%27
NIB0.25x500
Nuclease-free water1212
total 2000
  • ReagentT4 DNA Ligase - 100,000 unitsNew England BiolabsCatalog #M0202L
  • ReagentProtector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001
  • ReagentSUPERaseIN RNase InhibitorThermo Fisher ScientificCatalog #AM2696
  • PIC
  • Reagent10% Triton X-100Merck MilliporeSigma (Sigma-Aldrich)
  • ReagentBovine Albumin Fraction V (7.5% solution)Thermo FisherCatalog #15260037
  • NIB
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932

block1 mix:
ABCD
Blocker_11 mM22 μM12.1 μL
T4 Ligation buffer10x2x110 μL
Nuclease-free water427.9 μL
550 μL
  • ReagentT4 DNA Ligase - 100,000 unitsNew England BiolabsCatalog #M0202L
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932

block2 mix:

ABCD
Blocker_21mM26.4μM14.52 μL
T4 Ligation buffer10x2x110 μL
Nuclease-free water425.48 μL
550 μL
  • ReagentT4 DNA Ligase - 100,000 unitsNew England BiolabsCatalog #M0202L
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932

block3 mix:
ABCD
Blocker_31mM23 μM12.65μL
Triton X10010%0.1%5.5μL
Nuclease-free water531.85μL
550μL
  • Reagent10% Triton X-100Merck MilliporeSigma (Sigma-Aldrich)
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932
Ligation mix: (Amount0.5 mL per sample) 
ABCD
ReagentsStockingWorking 1x/μL
T4 ligation buffer10x1x50
RNase Inhibitor40 U/μL0.32 U/μl4
Superase RI20 U/μL0.05 U/μL1.25
PIC100x1x5
T4 DNA ligase2000 U/μL20 U/μl5
Triton X-10010% (vol/vol)0.1%5
BSA7.5%0.1%6.7
NIB0.2x100
Nuclease-free water323.05
total500
  • T4 ligation buffer
  • ReagentProtector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001
  • ReagentSUPERaseIN RNase InhibitorThermo Fisher ScientificCatalog #AM2696
  • PIC
  • ReagentT4 DNA Ligase - 100,000 unitsNew England BiolabsCatalog #M0202L
  • Reagent10% Triton X-100Merck MilliporeSigma (Sigma-Aldrich)
  • ReagentBovine Albumin Fraction V (7.5% solution)Thermo FisherCatalog #15260037
  • NIB
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932


Split-pool barcoding

Prepare 1% BSA in DPBS for rinsing plasticware, which helps to reduce sample loss.
Take out pre-prepared annealed linker-barcodes 96 well plates Go to , spin at Centrifigation700 x g, Room temperature, 00:02:00 , thaw and bring to TemperatureRoom temperature

2m
Re-suspend ConA-bead bound nuclei with Amount2 mL hybridisation mix for 48 barcodes in each round
Round 1
  • Add Amount40 µL of nuclei per well to linker1-BC1 plate , Centrifigation300 rpm, Room temperature, 00:30:00 , incubate on thermomixer (or Temperature23 °C )
  • Use multichannel pipettor to add Amount10 µL block1, pipetting up and down to mix,Centrifigation300 rpm, Room temperature, 00:30:00 , incubate on thermomixer (or Temperature23 °C )
  • Centrifuge Centrifigation300 x g, 00:02:00
  • Use multichannel pipette (rinsed with 1% BSA in DPBS) to collect cells into reservoir (rinsed with 1% BSA in DPBS)
1h 2m
Round 2
  • Add Amount55 µL of nuclei per well to linker2-BC2 plate, Centrifigation300 rpm, Room temperature, 00:30:00 , incubate on thermomixer
  • Use multichannel pipette to add Amount10 µL block2, pipetting up and down to mix,Centrifigation300 rpm, Room temperature, 00:30:00 , incubate on thermomixer (or Temperature23 °C )
  • Centrifuge Centrifigation300 x g, 00:02:00
  • Use multichannel pipette (rinsed tips with 1% BSA in DPBS) to collect nuclei into reservoir (also pre-rinsed with 1% BSA in DPBS)
1h 2m
Round 3
  • Add Amount70 µL of nuclei per well to linker3-BC3 plate, Centrifigation300 rpm, Room temperature, 00:30:00 , incubate on thermomixer (or Temperature23 °C )
  • Use multichannel pipette to add Amount10 µL block3, pipetting up and down to mix
  • Centrifuge Centrifigation300 x g, 00:02:00
  • Use multichannel pipette (rinsed tips with 1% BSA in DPBS) to collect nuclei into reservoir (also pre-rinsed with 1% BSA in DPBS) 
32m
Transfer nuclei suspension to Amount5 mL tube, centrifuge Centrifigation600 x g, 4°C, 00:03:00 , remove and discard the supernatant
3m
Transfer the pellet into a new Amount1.5 mL protein lobind tube , wash twice with Amount0.5 mL NIB (no RI, PIC), invert to mix in between

Ligation:

Re-suspend nuclei with Amount0.5 mL Ligation buffer, Centrifigation300 rpm, 25°C, 00:30:00 , incubate on thermomixer
30m
Briefly spin, and wash with Amount0.5 mL NIB (no RI, PIC),
re-suspend inAmount0.5 mL NIB (no RI, PIC),
filter through Amount30 µL strainer,
rinse strainer with Amount0.5 mL NIB
(Optional) Put strainer on top of a new tube, centrifuge Centrifigation600 x g, 4°C, 00:03:00
3m
Optional
Collect all nuclei into one tube, manually count nuclei by labelling nuclei with trypan blue (ReagentTrypan Blue Solution, 0.4%Thermo FisherCatalog #15250061 )

Based on estimated collision rate, aliquot desired number of nuclei into each sub-library.
Reverse crosslinking and lysis
1h
Reversing the light fixation performed in Go to

Equipment:
  • PCR machine

Preparing
2x reverse cross-linking buffer:
ABCD
ReagentsStockingIn buffer/μL
Tris pH 8.01M 100 mM100
NaCl5M 100 mM20
SDS10% 0.04%4
Nuclease-free water876
total1000
  • Reagent1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
  • ReagentSodium Chloride (5M)Invitrogen - Thermo FisherCatalog #AM9760G
  • ReagentSDS, 10% Solution, RNase-freeThermo FisherCatalog #AM9822
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932
Reverse crosslinking and lysis
Add NIB (Go to Nuclei Isolation Buffer ) to each sample to bring the volume to Amount20 µL in total

Amount20 µL of 2× reverse cross-linking buffer, Amount2 µL of Concentration20 mg/mL proteinase K (ReagentProteinase KThermo Fisher ScientificCatalog #EO0491 Concentration1 µg/µL in final), and Amount1.6 µL of RI mix (SUPERase RI:Protector 1:1) were mixed with each sample and incubated at Temperature55 °C for Duration01:00:00 in PCR machine 

1h
OPTIONAL STOP POINT
After adding lysis buffer, sub-libraries can be stored at Temperature-20 °C .

Optional
Pause
Add Amount2.5 µL of Concentration100 millimolar (mM) ReagentPMSFMerck MilliporeSigma (Sigma-Aldrich)Catalog #P7626 to the reverse crosslinked sample to inactivate proteinase K and incubate at TemperatureRoom temperature for Duration00:10:00 .  

10m
Attach to magnet and transfer supernatant into a new tube to remove ConA beads.
Myone C1 Streptavidin beads to separate tagged cDNA and gDNA
1h



Equipment:
  • end-to-end-rotator

Wash ReagentDynabeads MyOne Streptavidin C1Invitrogen - Thermo FisherCatalog #65001 (Amount10 µL for each sample, total 10 x N* μL) with Amount800 µL 1x B&W-T buffer three times, and re-suspend in Amount45 µL 2x Binding & Washing buffer + Amount1 µL ReagentSUPERaseIN RNase InhibitorThermo Fisher ScientificCatalog #AM2696 . 

*where N is the number of samples
Add Amount46 µL re-suspended beads into each lysed sample (Amount45.6 µL ), mixed and rotated on an end-to-end rotator at Centrifigation10 rpm, Room temperature, 01:00:00 , end-to-end rotator
1h
Put tubes on a magnetic stand to separate supernatant (transposed DNA fragments) and beads (cDNA). 
RNA library preparation
3h 12m
Equipment:
  • Qubit fluorometer
  • PCR machine (thermocycler)
  • end-to-end-rotator

Primers
AB
NameSequence
TSOAAGCAGTGGTATCAACGCAGAGTGAATrGrG+G
PA_FCAGACGTGTGCTCTTCCGATCT
PA_RAAGCAGTGGTATCAACGCAGAGT
P7_#1CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
P7_#2CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
P7_#3CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
P7_#4CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
P7_#5CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
P7_#6CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
P7_#7CAAGCAGAAGACGGCATACGAGATTTGACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
P7_#8CAAGCAGAAGACGGCATACGAGATGGAACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
P5_#1AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTAT
P5_#2AATGATACGGCGACCACCGAGATCTACACCTCTCTATTCGTCGGCAGCGTCAGATGTGTAT
P5_#3AATGATACGGCGACCACCGAGATCTACACTATCCTCTTCGTCGGCAGCGTCAGATGTGTAT
P5_#4AATGATACGGCGACCACCGAGATCTACACAGAGTAGATCGTCGGCAGCGTCAGATGTGTAT
P5_#5AATGATACGGCGACCACCGAGATCTACACGTAAGGAGTCGTCGGCAGCGTCAGATGTGTAT
P5_#6AATGATACGGCGACCACCGAGATCTACACACTGCATATCGTCGGCAGCGTCAGATGTGTAT
P5_#7AATGATACGGCGACCACCGAGATCTACACAAGGAGTATCGTCGGCAGCGTCAGATGTGTAT
P5_#8AATGATACGGCGACCACCGAGATCTACACCTAAGCCTTCGTCGGCAGCGTCAGATGTGTAT

Preparing
1x B&W-T-RI buffer:
  • Amount1 mL B&W-T buffer (Go to B&W-T buffer )
  • Amount5 µL ReagentSUPERaseIN RNase InhibitorThermo Fisher ScientificCatalog #AM2696 .
(Amount300 µL per sub-library) 

STE-RI buffer:
  • Amount1 mL STE buffer (Go to STE buffer )
  • Amount5 µL ReagentSUPERaseIN RNase InhibitorThermo Fisher ScientificCatalog #AM2696 .
(Amount100 µL per sub-library) 

Template switch mix:
ABCD
Reagent Stocking Working 1x 1x /μL
RT buffer5x 1x 10
Ficoll PM-400 20 % 4 % 10
PEG 6000 50 % 15 % 15
TSO primer100 μM 2.5 μM 1.25
dNTPs 25 mM/each 1 mM 2
RNAse Protector 40 U/μL 0.5 U/μL 0.625
Superase RI 20 U/μL 0.25 U/μL 0.625
Maxima H Minus RT200 U/μL 10 U/μl 2.5
Nuclease-free water  8
  total 50
  • RT buffer (Maxima H minus RT)
  • ReagentFicoll PM‐400Merck MilliporeSigma (Sigma-Aldrich)Catalog #F5415-50ML
  • ReagentPEG 6000 (Poly(ethylene glycol))Bio Basic Inc.Catalog #PB0432.SIZE.500g
  • TSO (Template Switching Optimised) primer
  • ReagentdNTP Set 100 mM SolutionsThermo Fisher ScientificCatalog #R0182
  • ReagentProtector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335402001
  • ReagentSUPERaseIN RNase InhibitorThermo Fisher ScientificCatalog #AM2696
  • ReagentMaxima H Minus Reverse TranscriptaseThermo Fisher ScientificCatalog ##EP0741
This is an optimised Moloney murine leukemia virus Reverse Transcriptase (M-MuLV RT) which has terminal transferase activity. This means it can perform non-template addition of nucleotides to 3'-ends with a preference for dCTP. The resulting poly C overhang allows the TSO primer, which ends in 3 rGs, to anneal. This RT is also capable of template switching, meaning that it can read through the gap in the backbone between the end of the RNA template and the annealed TSO and continue elongating with the TSO as the template.
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932

RNA library preparation

Wash beads three times using Amount100 µL 1x B&W-T buffer with ReagentSUPERaseIN RNase InhibitorThermo Fisher ScientificCatalog #AM2696 Amount0.1 µL mix by inverting between washes

Wash beads once using Amount100 µL STE buffer with ReagentSUPERaseIN RNase InhibitorThermo Fisher ScientificCatalog #AM2696 Amount0.1 µL while tube on magnet, without re-suspending beads

Re-suspend beads with Amount50 µL Template switch mix by pipetting
Rotate beads Centrifigation10 rpm, Room temperature, 00:30:00 , end-to-end-rotator
Re-suspend by pipetting
Incubate for Duration01:30:00 at Temperature42 °C in PCR machine

2h
Pre-Amplification
After template switching, add Amount100 µL of STE to each tube to dilute the sample.
Remove the supernatant by placing the sample on a magnetic stand.
Wash beads with Amount200 µL of STE without disturbing the bead pellet
Beads were then re-suspended in Amount55 µL of PCR mix:  
ABCD
Reagent Stocking Working 1x 1x /μL
Kapa HiFi PCR mix 2x 1x 27.5
PA_F10 μM 400 nM 2.2
PA_R10 μM 400 nM 2.2
Nuclease-free water  23.1
  total 55
  • ReagentHotStart ReadyMix (KAPA HiFi PCR kit)Kapa BiosystemsCatalog #KK2601
  • PA_F & PA_R primers
  • ReagentNuclease-free water, not DEPC-treated Life TechnologiesCatalog #AM9932

Run following programme to pre-amplify cDNA:
- Temperature95 °C Duration00:03:00  
- 12 cycles of:
- Temperature98 °C Duration00:00:30
- Temperature65 °C Duration00:00:45
- Temperature72 °C Duration00:03:00
- Temperature72 °C Duration00:05:00  
- Temperature4 °C Hold 
12m 15s
Purify by 0.8x Reagent Ampure XP beads Beckman CoulterCatalog #A63881 and elute in Amount32 µL 0.1x EB (Elution Buffer)

Measure concentration with Qubit

Note
Libraries made from ~1000-5000 cells and amplified for 12-15 cycles typically yield around Amount500 ng after purification

Tagging MEA and PCR amplification
Setup following reaction; incubate in PCR machine at Temperature37 °C for Duration00:30:00  
AB
1:20 pA-Tn5-MeA-MeR2 μl
4x Tag Buffer5 μl
Sample Volume to give 50ng
Nuclease-free waterTo 20 μl

30m
Add Amount2 µL 0.2% SDS mix and incubate at TemperatureRoom temperature for Duration00:10:00 to release tagged fragments; neutralise SDS with Amount1 µL 4% Triton X100

  • ReagentSDS, 10% Solution, RNase-freeThermo FisherCatalog #AM9822
  • Reagent10% Triton X-100Merck MilliporeSigma (Sigma-Aldrich)

10m
Amplify libraries with NEBnext mix as following 
AB
Sample23 μl
2x NEBnext mix25 μl
P5_#N 25μM1 μl
P7_#N 25uM 1 μl
Run the following programme to amplify libraries:  
- Temperature72 °C Duration00:10:00
- Temperature98 °C Duration00:03:00
- 11 cycles of:
- Temperature98 °C Duration00:00:10
- Temperature65 °C Duration00:00:30
- Temperature72 °C Duration00:01:00
- Temperature72 °C Duration00:05:00
- Temperature4 °C hold 
19m 45s
Purify the amplified library using 0.7x Reagent Ampure XP beads Beckman CoulterCatalog #A63881 three times and elute with Amount32 µL of EB.
DNA library amplification
15m 40s
Equipment:
  • PCR machine (thermocycler)
  • Qubit fluorometer
  • bioanalyzer

Primers
In addition to the P7_#N and P5_#N primers listed above in the RNA library prep section you will need:

AB
NameSequence
MEA_LNATCGTCGGCAGCGTC AGATGTGTA+TA+AG+AG+AC+AG/3InvdT/


Purify the DNA component using 1.2x Reagent Ampure XP beads Beckman CoulterCatalog #A63881 , elute with Amount21 µL 0.1x EB buffer 
Add MEA by adapter switching


Modified from Mulqueen et al. 2021 "[symmetrical strand single-cell combinatorial indexing (s3)]
uses single-adapter transposition to incorporate the forward primer sequence, the Tn5 mosaic end sequence and a reaction-specific DNA barcode. As with standard tagmentation workflows, extension through the bottom strand is then performed to provide adaptor sequences on both ends of each molecule; however, the s3 transposome complexes contain a uracil base immediately following the mosaic end sequence. Use of a uracil-intolerant polymerase therefore prevents extension beyond the mosaic end into the DNA barcode and forward adaptor sequence. A second template oligo is then introduced that contains a 3’-blocked locked nucleic acid (LNA) mosaic end reverse complement sequence with a reverse adaptor sequence 5’ overhang. This oligo favorably anneals to the copied mosaic end sequence, due to the higher melting temperature of LNA, and acts as a template for the library molecule to extend through and copy the reverse adaptor. This results in all library fragments having both a forward and reverse adaptor sequence. The LNA-templated extension is carried out over multiple rounds of thermocycling to ensure maximum efficiency of reverse adaptor incorporation."

Citation
Mulqueen RM, Pokholok D, O'Connell BL, Thornton CA, Zhang F, O'Roak BJ, Link J, Yardımcı GG, Sears RC, Steemers FJ, Adey AC (2021). High-content single-cell combinatorial indexing.
https://doi.org/10.1038/s41587-021-00962-z
LINK

Gap filling reaction

Amount20 µL sample (use all of the sample) + Amount8 µL ReagentNEBNext® High-Fidelity 2X PCR Master MixNew England BiolabsCatalog #M0541
run: Temperature72 °C Duration00:10:00  
10m
Adapter switching reaction

Add Amount3 µL Concentration1 micromolar (µM) MEA_LNA 
  • run: Temperature98 °C Duration00:00:30
  • 10 cycles of :
- Temperature98 °C Duration00:00:10
- Temperature59 °C Duration00:00:20
- Temperature72 °C Duration00:00:10
1m 10s
Amplification reaction

Add:
  • Amount50 µL Q5U 2x readymix DNA polymerase
  • Amount15 µL H2O
  • Amount2 µL P7_#N (Concentration25 micromolar (µM) ) 
  • Amount2 µL P5_#N (Concentration25 micromolar (µM) ) 

run: Temperature98 °C Duration00:00:30
  • 10 cycles of:
- Temperature98 °C Duration00:00:10
- Temperature55 °C Duration00:00:20
- Temperature72 °C Duration00:00:30
  • Temperature72 °C Duration00:03:00
  • Temperature4 °C hold 
4m 30s
Take out Amount50 µL into a new tube and store at Temperature-20 °C as a backup, and add extra cycles (4-10 cycles, depending on the sample) to the remaining libraries to generate enough material for sequencing. 
Purify libraries with 0.7x Reagent Ampure XP beads Beckman CoulterCatalog #A63881 twice, elute with Amount32 µL EB, check concentration with Qubit, and run samples on Bioanalyzer. 
Sequencing
Use a 150 bp paired-end sequencing kit with the following customised parameters:

  • Read 1 with Nextera adapter for 100 bp. (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG)
  • Read 2 with Illumina Truseq adapter for 200 bp. (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT)
  • Read 2 contains 128 bp for cell barcodes and UMI sequences, plus 72 bp for genomic sequence.
  • i7 index is 6bp
  • i5 index is 8bp

Normally, we add 10% PhiX spike-in for Illumina sequencing machines, and 2% PhiX spike-in for Element AVITI machine.

Data Processing & Analysis
Data processing can be performed with our analysis pipeline.
See the README file in the pipeline repository for additional details.
Software
nf_mtr_seq
NAME
https://github.com/Rugg-Gunn-Lab/nf_mtr_seq
REPOSITORY
https://doi.org/10.5281/zenodo.1504690
SOURCE LINK

Protocol references
3XFlag-pATn5: https://dx.doi.org/10.17504/protocols.io.8yrhxv6
CUT&Tag: https://doi.org/10.1038/s41467-019-09982-5
Split-seq: https://www.science.org/doi/10.1126/science.aam8999
Split-seq protocol: https://www.seeliglab.org/uploads/5/9/9/7/59974251/split-seq_protocol_v3.0
Share-seq: https://doi.org/10.1016/j.cell.2020.09.056
Paired-seq: https://doi.org/10.1038/s41594-019-0323-x
Paired-Tag: https://doi.org/10.1038/s41592-021-01060-3
Adapter switch S3: https://doi.org/10.1038/s41587-021-00962-z
Citations
Step 26
Mulqueen RM, Pokholok D, O'Connell BL, Thornton CA, Zhang F, O'Roak BJ, Link J, Yardımcı GG, Sears RC, Steemers FJ, Adey AC. High-content single-cell combinatorial indexing.
https://doi.org/10.1038/s41587-021-00962-z