Jan 07, 2025

Public workspaceSpatial Multi-omics Sequencing for Formalin-Fixed Paraffin Embedded Tissue via DBiT-seq

 Forked from Spatial Multi-omics Sequencing for Fixed Tissue via DBiT-seq
DOI
https://dx.doi.org/10.17504/protocols.io.261ger2wwl47/v1
Spatial Multi-omics Sequencing for Formalin-Fixed Paraffin Embedded Tissue via DBiT-seq
  • Archibald Enninful1,
  • Rong Fan1,
  • Negin Farzad1
  • 1Yale University
  • Fan Lab Yale
Archibald Enninful
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DOI: https://dx.doi.org/10.17504/protocols.io.261ger2wwl47/v1
External link: https://www.cell.com/cell/fulltext/S0092-8674(24)01019-5
Protocol Citation: Archibald Enninful, Rong Fan, Negin Farzad 2025. Spatial Multi-omics Sequencing for Formalin-Fixed Paraffin Embedded Tissue via DBiT-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.261ger2wwl47/v1
Manuscript citation:
Bai et al., 2024, Cell 187, 6760–6779
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: January 06, 2025
Last Modified: January 07, 2025
Protocol Integer ID: 117754
Keywords: cell resolution for spatial transcriptomic, spatial transcriptomic, tissue for spatial omic, rna biology in archival formalin, fixed paraffin embedded tissue via dbit, ffpe tissue sample, embedded tissue, fixed paraffin embedded tissue, exploring rna biology, rna, single cell, spatial omic, combinatorial dna oligo, cell resolution, omics atlas, tissue
Funders Acknowledgements:
SenNet Consortium
Grant ID: 1U54AG076043-01
Abstract
This protocol describes the use of Deterministic Barcoding in Tissue for spatial omics sequencing (DBiT-seq) platform to construct a multi-omics atlas on FFPE tissue samples. This approach uses a microfluidic-based method to introduce combinatorial DNA oligo barcodes directly to the cells in a tissue section on a glass slide. This technique does not directly resolve single cells but can achieve a near-single-cell resolution for spatial transcriptomics.

This protocol is adapted from the Patho-DBiT publication - Spatially exploring RNA biology in archival formalin fixed paraffin-embedded tissues (Bai et al., 2024, Cell 187, 6760–6779)
Image Attribution
Image from graphical abstract of this publication - Bai et al., 2024, Cell 187, 6760–6779
Materials
KEY RESOURCES TABLE
REAGENT or RESOURCESOURCEIDENTIFIER
Antibodies
TotalSeqTM antibodies Biolegend See Table 1
Chemicals, Peptides, and Recombinant Proteins
Maxima H Minus Reverse Transcriptase (200 U/L) Thermo Fisher Scientific EP0751
dNTP mix Thermo Fisher Scientific R0192
RNase Inhibitor Enzymatics Y9240L
SUPERase• InTM RNase Inhibitor Thermo Fisher Scientific AM2694
T4 DNA Ligase New England Biolabs M0202L
KAPA Pure Beads Kapa Biosystems KK8002
DynabeadsTM MyOneTM Streptavidin C1 Thermo Fisher Scientific 65001
Proteinase K, recombinant, PCR grade Thermo Fisher Scientific EO0491
Kapa HiFi Hotstart ReadyMix PCR Kit Kapa Biosystems KK2601
Formaldehyde solution Sigma F8775-25ML
NEBuffer 3.1 New England Biolabs B7203S
T4 DNA Ligase Reaction Buffer New England Biolabs B0202S
Evagreen Dye, 20X in water Biotium 31000-T
Oligonucleotides
Primers, Ligation linkers, DNA barcodes IDT See Table 2
Critical Commercial Assays
DNA Clean & Concentrator with Zymo-Spin I Columns Research Products International ZD4004
Nextera XT DNA Preparation Kit Illumina FC-131-1024
Other
Multiplate PCR Plate, 96-well, clear BioRad MLL9601
50 mL Falcon Tube Corning 352070
BioDot Pure 8 – Strip PCR tubes w/ Optically Clear Flat Caps Dot Scientific Inc. 403-8PCR
SYLGARDTM 184 Silicone Elastomer Base and Curing Agent Dow Corning 4019862
SuperChipTM Poly-L-Lysine Slides Electron Microscopy Sciences 63478-AS
RNaseZapTM RNase Decontamination Solution Thermo Fisher Scientific AM9780
Silicon wafer WaferPro C04004
Photoresist SU-8 2010 MicroChem Laboratory SU-8 2010
CategoryBarcodeSpecificityCloneBarcode Sequence
TotalSeqTM-A12CD117 (c-kit)2B8TGCATGTCATCGGTG
TotalSeqTM-A78CD49dR1-2CGCTTGGACGCTTAA
TotalSeqTM-A96CD4530-F11TGGCTATGGAGCAGA
TotalSeqTM-A104CD1023C4 (MIC2/4)GATATTCAGTGCGAC
TotalSeqTM-A115FcεRIαMAR-1AGTCACCTCGAAGCT
TotalSeqTM-A118NK-1.1avas12GTAACATTACTCGTC
TotalSeqTM-A119Siglec H551CCGCACCTACATTAG
TotalSeqTM-A122TER-119/Erythroid CellsTER-119GCGCGTTTGTGCTAT
TotalSeqTM-A130Ly-6A/E (Sca-1)D7TTCCTTTCCTACGCA
TotalSeqTM-A232MAdCAM-1MECA-367TTGGGCGATTAAGAA
TotalSeqTM-A381Panendothelial Cell AntigenMECA-32CGTCCTAGTCATTGG
TotalSeqTM-A415P2RY12S16007DTTGCTTATTTCCGCA
TotalSeqTM-A439CD201 (EPCR)RCR-16TATGATCTGCCCTTG
TotalSeqTM-A442Notch 1HMN1-12TCCGGTCACTCAGTA
TotalSeqTM-A443CD41MWReg30ACTTGGATGGACACT
TotalSeqTM-A449CD326 (Ep-CAM)G8.8ACCCGCGTTAGTATG
TotalSeqTM-A552CD304 (Neuropilin-1)3E12CCAGCTCATTCAACG
TotalSeqTM-A553CD309 (VEGFR2, Flk-1)Avas12ATAAGAGCCCACCAT
TotalSeqTM-A558CD55 (DAF)RIKO-3ATTGTTGTCAGACCA
TotalSeqTM-A559CD63NVG-2ATCCGACACGTATTA
TotalSeqTM-A564Folate Receptor β (FR-β)10/FR2CTCAGATGCCCTTTA
TotalSeqTM-A596ESAM1G8/ESAMTATAGTTTCCGCCGT
Table S1. TotalSeqTMAntibodies, related to Step 9.

Oligo NameSequence
Primer 1CAAGCGTTGGCTTCTCGCATCT
Primer 2AAGCAGTGGTATCAACGCAGAGT
Primer 3 (cite-seq)CCTTGGCACCCGAGAATT*C*C
Ligation LinkerCGAATGCTCTGGCCTCTCAAGCACGTGGAT
Template Switch PrimerAAGCAGTGGTATCAACGCAGAGTGAATrGrG+G
P5 oligoAATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
P5 oligo (cite-seq)AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTTGGCACCCGAGAATTCCA
P7 oligo (701)CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT
P7 oligo (702)CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT
P7 oligo (703)CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT
P7 oligo (704)CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT
P7 oligo (705)CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT
P7 oligo (706)CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAGCGTTGGCTTCTCGCATCT
Table S2A. List of PCR Oligo.
AB
Barcode A-1/5Phos/AGGCCAGAGCATTCGAACGTGATTTTTTTTTTTTTTTTVN
Barcode A-2/5Phos/AGGCCAGAGCATTCGAAACATCGTTTTTTTTTTTTTTTVN
Barcode A-3/5Phos/AGGCCAGAGCATTCGATGCCTAATTTTTTTTTTTTTTTVN
Barcode A-4/5Phos/AGGCCAGAGCATTCGAGTGGTCATTTTTTTTTTTTTTTVN
Barcode A-5/5Phos/AGGCCAGAGCATTCGACCACTGTTTTTTTTTTTTTTTTVN
Barcode A-6/5Phos/AGGCCAGAGCATTCGACATTGGCTTTTTTTTTTTTTTTVN
Barcode A-7/5Phos/AGGCCAGAGCATTCGCAGATCTGTTTTTTTTTTTTTTTVN
Barcode A-8/5Phos/AGGCCAGAGCATTCGCATCAAGTTTTTTTTTTTTTTTTVN
Barcode A-9/5Phos/AGGCCAGAGCATTCGCGCTGATCTTTTTTTTTTTTTTTVN
Barcode A-10/5Phos/AGGCCAGAGCATTCGACAAGCTATTTTTTTTTTTTTTTVN
Barcode A-11/5Phos/AGGCCAGAGCATTCGCTGTAGCCTTTTTTTTTTTTTTTVN
Barcode A-12/5Phos/AGGCCAGAGCATTCGAGTACAAGTTTTTTTTTTTTTTTVN
Barcode A-13/5Phos/AGGCCAGAGCATTCGAACAACCATTTTTTTTTTTTTTTVN
Barcode A-14/5Phos/AGGCCAGAGCATTCGAACCGAGATTTTTTTTTTTTTTTVN
Barcode A-15/5Phos/AGGCCAGAGCATTCGAACGCTTATTTTTTTTTTTTTTTVN
Barcode A-16/5Phos/AGGCCAGAGCATTCGAAGACGGATTTTTTTTTTTTTTTVN
Barcode A-17/5Phos/AGGCCAGAGCATTCGAAGGTACATTTTTTTTTTTTTTTVN
Barcode A-18/5Phos/AGGCCAGAGCATTCGACACAGAATTTTTTTTTTTTTTTVN
Barcode A-19/5Phos/AGGCCAGAGCATTCGACAGCAGATTTTTTTTTTTTTTTVN
Barcode A-20/5Phos/AGGCCAGAGCATTCGACCTCCAATTTTTTTTTTTTTTTVN
Barcode A-21/5Phos/AGGCCAGAGCATTCGACGCTCGATTTTTTTTTTTTTTTVN
Barcode A-22/5Phos/AGGCCAGAGCATTCGACGTATCATTTTTTTTTTTTTTTVN
Barcode A-23/5Phos/AGGCCAGAGCATTCGACTATGCATTTTTTTTTTTTTTTVN
Barcode A-24/5Phos/AGGCCAGAGCATTCGAGAGTCAATTTTTTTTTTTTTTTVN
Barcode A-25/5Phos/AGGCCAGAGCATTCGAGATCGCATTTTTTTTTTTTTTTVN
Barcode A-26/5Phos/AGGCCAGAGCATTCGAGCAGGAATTTTTTTTTTTTTTTVN
Barcode A-27/5Phos/AGGCCAGAGCATTCGAGTCACTATTTTTTTTTTTTTTTVN
Barcode A-28/5Phos/AGGCCAGAGCATTCGATCCTGTATTTTTTTTTTTTTTTVN
Barcode A-29/5Phos/AGGCCAGAGCATTCGATTGAGGATTTTTTTTTTTTTTTVN
Barcode A-30/5Phos/AGGCCAGAGCATTCGCAACCACATTTTTTTTTTTTTTTVN
Barcode A-31/5Phos/AGGCCAGAGCATTCGGACTAGTATTTTTTTTTTTTTTTVN
Barcode A-32/5Phos/AGGCCAGAGCATTCGCAATGGAATTTTTTTTTTTTTTTVN
Barcode A-33/5Phos/AGGCCAGAGCATTCGCACTTCGATTTTTTTTTTTTTTTVN
Barcode A-34/5Phos/AGGCCAGAGCATTCGCAGCGTTATTTTTTTTTTTTTTTVN
Barcode A-35/5Phos/AGGCCAGAGCATTCGCATACCAATTTTTTTTTTTTTTTVN
Barcode A-36/5Phos/AGGCCAGAGCATTCGCCAGTTCATTTTTTTTTTTTTTTVN
Barcode A-37/5Phos/AGGCCAGAGCATTCGCCGAAGTATTTTTTTTTTTTTTTVN
Barcode A-38/5Phos/AGGCCAGAGCATTCGCCGTGAGATTTTTTTTTTTTTTTVN
Barcode A-39/5Phos/AGGCCAGAGCATTCGCCTCCTGATTTTTTTTTTTTTTTVN
Barcode A-40/5Phos/AGGCCAGAGCATTCGCGAACTTATTTTTTTTTTTTTTTVN
Barcode A-41/5Phos/AGGCCAGAGCATTCGCGACTGGATTTTTTTTTTTTTTTVN
Barcode A-42/5Phos/AGGCCAGAGCATTCGCGCATACATTTTTTTTTTTTTTTVN
Barcode A-43/5Phos/AGGCCAGAGCATTCGCTCAATGATTTTTTTTTTTTTTTVN
Barcode A-44/5Phos/AGGCCAGAGCATTCGCTGAGCCATTTTTTTTTTTTTTTVN
Barcode A-45/5Phos/AGGCCAGAGCATTCGCTGGCATATTTTTTTTTTTTTTTVN
Barcode A-46/5Phos/AGGCCAGAGCATTCGGAATCTGATTTTTTTTTTTTTTTVN
Barcode A-47/5Phos/AGGCCAGAGCATTCGCAAGACTATTTTTTTTTTTTTTTVN
Barcode A-48/5Phos/AGGCCAGAGCATTCGGAGCTGAATTTTTTTTTTTTTTTVN
Barcode A-49/5Phos/AGGCCAGAGCATTCGGATAGACATTTTTTTTTTTTTTTVN
Barcode A-50/5Phos/AGGCCAGAGCATTCGGCCACATATTTTTTTTTTTTTTTVN
Table S2B. List of DNA Barcode A Sequences.
AB
Barcode B-1/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAACGTGATATCCACGTGCTTGAG
Barcode B-2/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAAACATCGATCCACGTGCTTGAG
Barcode B-3/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNATGCCTAAATCCACGTGCTTGAG
Barcode B-4/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAGTGGTCAATCCACGTGCTTGAG
Barcode B-5/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACCACTGTATCCACGTGCTTGAG
Barcode B-6/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACATTGGCATCCACGTGCTTGAG
Barcode B-7/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCAGATCTGATCCACGTGCTTGAG
Barcode B-8/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCATCAAGTATCCACGTGCTTGAG
Barcode B-9/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCGCTGATCATCCACGTGCTTGAG
Barcode B-10/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACAAGCTAATCCACGTGCTTGAG
Barcode B-11/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCTGTAGCCATCCACGTGCTTGAG
Barcode B-12/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAGTACAAGATCCACGTGCTTGAG
Barcode B-13/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAACAACCAATCCACGTGCTTGAG
Barcode B-14/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAACCGAGAATCCACGTGCTTGAG
Barcode B-15/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAACGCTTAATCCACGTGCTTGAG
Barcode B-16/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAAGACGGAATCCACGTGCTTGAG
Barcode B-17/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAAGGTACAATCCACGTGCTTGAG
Barcode B-18/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACACAGAAATCCACGTGCTTGAG
Barcode B-19/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACAGCAGAATCCACGTGCTTGAG
Barcode B-20/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACCTCCAAATCCACGTGCTTGAG
Barcode B-21/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACGCTCGAATCCACGTGCTTGAG
Barcode B-22/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACGTATCAATCCACGTGCTTGAG
Barcode B-23/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNACTATGCAATCCACGTGCTTGAG
Barcode B-24/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAGAGTCAAATCCACGTGCTTGAG
Barcode B-25/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAGATCGCAATCCACGTGCTTGAG
Barcode B-26/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAGCAGGAAATCCACGTGCTTGAG
Barcode B-27/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNAGTCACTAATCCACGTGCTTGAG
Barcode B-28/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNATCCTGTAATCCACGTGCTTGAG
Barcode B-29/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNATTGAGGAATCCACGTGCTTGAG
Barcode B-30/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCAACCACAATCCACGTGCTTGAG
Barcode B-31/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNGACTAGTAATCCACGTGCTTGAG
Barcode B-32/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCAATGGAAATCCACGTGCTTGAG
Barcode B-33/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCACTTCGAATCCACGTGCTTGAG
Barcode B-34/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCAGCGTTAATCCACGTGCTTGAG
Barcode B-35/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCATACCAAATCCACGTGCTTGAG
Barcode B-36/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCCAGTTCAATCCACGTGCTTGAG
Barcode B-37/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCCGAAGTAATCCACGTGCTTGAG
Barcode B-38/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCCGTGAGAATCCACGTGCTTGAG
Barcode B-39/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCCTCCTGAATCCACGTGCTTGAG
Barcode B-40/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCGAACTTAATCCACGTGCTTGAG
Barcode B-41/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCGACTGGAATCCACGTGCTTGAG
Barcode B-42/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCGCATACAATCCACGTGCTTGAG
Barcode B-43/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCTCAATGAATCCACGTGCTTGAG
Barcode B-44/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCTGAGCCAATCCACGTGCTTGAG
Barcode B-45/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCTGGCATAATCCACGTGCTTGAG
Barcode B-46/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNGAATCTGAATCCACGTGCTTGAG
Barcode B-47/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNCAAGACTAATCCACGTGCTTGAG
Barcode B-48/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNGAGCTGAAATCCACGTGCTTGAG
Barcode B-49/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNGATAGACAATCCACGTGCTTGAG
Barcode B-50/5Biosg/CAAGCGTTGGCTTCTCGCATCTNNNNNNNNNNGCCACATAATCCACGTGCTTGAG
Table S2C. List of DNA Barcode B Sequences.


  • This protocol requires using a microfluidic device fabricated with PDMS using soft lithography.

  • A hole punching machine (SCHMIDT® Manual Press) is needed to punch the inlet and outlet holes in the PDMS chip after its fabrication.

  • The tissue of interest should be placed on the center of a poly-L-lysine coated glass slide. (CatLog no. 63478-AS, electron microscopy sciences).

  • A custom-designed acrylic clamp with screws is needed to hold the PDMS device and the glass slide together firmly.

  • The silicon wafer used for fabricating the PDMS mold is purchased from WaferPro (CatLog. No. C04004).

  • The photoresist is purchased from MicroChem Laboratory (CatLog. No. SU-8 2010).

  • A homemade laboratory vacuum system(See Figure Below) is needed to applying suction to move fluid in the microfluidic channels.

  • Microscopy – The tissue of interest can be scanned and imaged using EVOS (Thermo Fisher EVOS fl), typically at a magnification of 10×. Any suitable optical microscope can be used.

  • A Laser Engraver Cutter is used to cut out the acrylic barcode and lysis clamps.

  • A “humidified chamber” (See Figure Below) to prevent reagent evaporation during incubation.

Key equipment used in DBiT-seq

Hole punch machine, the key device for DBiT-seq, and the house vacuum lines. (Left) SCHMIDT® Manual Press used for hole punching 2-mm diameter holes for the inlets and outlets of Chip-A and Chip-B. (Top Right) Example of a “humidified chamber” to prevent reagent evaporation during the incubation steps. (Bottom Right) House vacuum used for pulling reagents through the microchannels.
Recipes

Critical: Prepare the RT mix, template switch mix, ligation mix, PCR mix, 1X lysis solution, flow wash buffer, PBS-RI, 80% ethanol on the same day as usage. 2X lysis buffer, 1X B&W Buffer with 0.05% Tween-20, and 2X B&W Buffer can be stored for up to 6 months at room temperature.

PBS-RI
ReagentVolume (μL)
1X PBS 5000
RNase Inhibitor (40 U/µL) (Enzymatics) 7
Total 5007
80% Ethanol
ReagentVolume(ml)
RNase-free water 1
100% Ethanol 4
Total 5
RT Mixture
ReagentVolume (μL)
5X Maxima RT buffer 50
RNase-free water 32.8
RNase Inhibitor (Enzymatics) 1.6
Superase In RNase Inhibitor (Ambion) 3.2
dNTPs (10 mM stock) 12.5
Maxima H Minus Reverse Transcriptase 25
PBS-RI 100
Total 225.1
Ligation Mix
ReagentVolume (μL)
10X T4 Ligase Buffer 27
1X NEB buffer 3.1 with 1% RI (Enzymatics) 115.8
5% Triton-X100 5.4
RNase Inhibitor (Enzymatics) 2.2
RNase-free water 69.5
SuperaseIn RNase Inhibitor (Ambion) 0.7
T4 DNA Ligase (400 U/µL) 11
Total 231.6
Template switch mix
ReagentVolume (μL)
20% Ficoll PM-400 44
5X Maxima RT buffer 44
dNTPs (10 mM stock) 22
RNase Inhibitor (Enzymatics) 5.5
Maxima H Minus Reverse Transcriptase 11
Template Switch Primer (100 μM stock) 5.5
RNase-free water 88
Total 220
PCR mix
ReagentVolume (μL)
2X Kapa Hifi HotStart master mix 110
Primer1 BC_0062 (10μM) 8.8
Primer2 BC_0108 (10μM) 8.8
RNase-free water 92.4
Total 220
Flow Wash Buffer
ReagentVolume (mL)
1X PBS 4
10 % Triton X-100 0.04
Superase In RNase Inhibitor 0.01
Total volume 4.05
1X Lysis Solution
ReagentVolume (μL)
1X PBS 50
2X Lysis Buffer 50
Proteinase K (20mg/mL) (Thermo) 10
Total 110
2X Lysis Buffer
ReagentStock ConcentrationFinal Concentration (2X)Volume (mL)
Tris, pH 8.0 1 M 20 mM 0.5
NaCl 5 M 400 mM 2
EDTA, pH 8.0 0.5 M 100 mM 5
SDS 10% 4.4 % 11
RNase-free Water NA NA 6.5
Final Volume 25
1X B&W Buffer with 0.05% Tween-20
ReagentVolume
1M Tris-HCl pH 8.0 100 μL
EDTA, 0.5M 20 μL
5M NaCl 4 mL
Tween 20 10% 100 μL
RNase-free water 15.78 mL
Total 20 mL
2X B&W Buffer
ReagentVolume
1M Tris-HCl pH 8.0 500 μL
EDTA, 0.5M 100 μL
5M NaCl 20 mL
RNase-free water 29.4 mL
Total 50 mL

CRITICAL: Handle all in an RNase-free area.

Troubleshooting
Before start
The protocol below describes the reagents, equipment, and specific experimental steps for using the DBiT-seq platform on frozen fixed tissue slides. The 10μm, 25μm, 50μm microfluidic channel width were designed and validated to provide spatial profiling at different resolutions.

CRITICAL: Work in an RNase-free environment when the microfluidic device is not on the tissue slide. Use RNaseZapTM or other commercially available cleaner solution and filter-tips. Clean surfaces and gloves with RNaseZapTM.

CRITICAL:Keep reagents on ice at all times.
Fabricating the Silicon Wafer Device Mold
12h
Prepare a high-resolution computer-aided-design (CAD) file with the desired microfluidics chip design. A CAD file is also available in the link provided. (https://ars.els-cdn.com/content/image/1-s2.0-S2666166721002392-mmc4.zip)
50Prepare chrome photomasks of the microfluidics chip by printing the high-resolution CAD files onto a glass substrate (Figure 1).

Figure 1: Microfluidic Photomask Design.
(Left) AutoCAD design for the 25 micron-width 50 channel Chip-A and Chip-B. (Right) AutoCAD design for 10 micron-width channel Chip-A and Chip-B.
NOTE: We have outsourced this step (Front Range Photomask, USA).

Using these masks, prepare a replica mold as follows:
Start the process by cleaning a 4-inch silicon wafer with 100% acetone (Aldrich) and then 100% isopropanol (Aldrich), then dry with compressed air.

NOTE: Acetone and isopropanol are mildly toxic. Use proper PPE when handling and discard waste in the appropriate containers.
Bake the wafer at 180°C for 10 min on a hot plate to dry it out.
Use a spin coater to evenly spread SU-8 2010 photoresist (MicroChem) for 10 μm device or SU-8 2025 (MicroChem) for 25 μm and 50 μm device onto the wafer at 500 rpm for 5 seconds followed by 1100 rpm (10 μm device), 3500 rpm (25 μm device) or 1750 rpm (50 μm device) for 40 seconds.
Soft bake the wafer for 3 min at 65°C (25 μm and 50 μm device) and 4 min (10 μm device) or 6 min (25 μm and 50 μm device) at 95°C.
Expose the SU-8 on the wafer through the photomask using Mask Aligner with a dose of 150 mJ/cm2 UV.
For the post exposure bake, bake the wafer for 1 min at 65°C (25 μm and 50 μm device) and 5 min (10 μm and 25 μm device) or 6 min (50 μm device) at 95°C.
Develop the SU-8 for 4 min (10 μm and 25 μm device) or 5 min (50 μm device) in a bath of ∼50 mL SU-8 developer (MicroChem).
Rinse the wafer with 100% isopropanol and dry with compressed air.
Perform a hard bake by baking the wafer for 10 min at 180°C.

CRITICAL: This process needs to be carried out in a microelectronics cleanroom.

Pause Point: The replica mold can now be stored and reused indefinitely.
Creating the Acrylic Clamps
30m
Prepare the pattern and dimensions for the barcoding clamp and lysis clamp.
Peel covering of acrylic sheet and place it in the laser cutting machine.
Select the program dimensions and cut two pieces for the top and bottom of the barcoding clamp.
Place the top blank piece into the laser cutter and select the pattern to cut out the four holes in the corner for the screws and nuts.
Remove cut-out scraps with a pipet tip or a similar pointed tool.
Repeat steps 15 and 16 for the bottom blank piece.
Repeat steps 13 to 17 for the lysis clamp using the correct dimensions and pattern with an additional hole in the center of the top piece (Example shown in Figure 2).

Figure 2. Key parts for setup. PDMS reservoirs and acrylic clamps used in DBiT-seq. (A) Inlet Reservoir. (B) Barcoding Clamp. (C) Lysis Reservoir. (D) Lysis Clamp.

NOTE: The acrylic clamps can be reused indefinitely.
Preparing the Microfluidic Device and reagent reservoirs
3h
Thoroughly mix polydimethylsiloxane (PDMS) elastomer base and curing agent (these come together) at a 10:1 ratio (See Figure 3).

Figure 3: Step-by-step visual guide for making Chip-A and Chip-B.

Pour the mixture into the silicon device mold.

NOTE: Aim for a chip height of about 5mm.
Place in a vacuum desiccator until all bubbles dissipate from the mixture about 30-60 min.
Cure in an oven at 65-70˚C for a minimum of 2 hours and up to overnight.

NOTE: Make sure to place on an even surface to prevent uneven curing!
Cut out the cured device and hole punch each of the 50 inlets and outlets for the channels.

NOTE: Cut both Chip-A and Chip-B in the approximate size of a glass slide.
NOTE: Make sure there are no PDMS pieces left in the inlets or outlets.
Thoroughly clean the surface of the device with scotch tape.
Pour mixture in a container large enough to cut out two roughly 25x25x5mm PDMS pieces.
For the barcoding reservoir, cut out a 20x20mm piece in the center, barely large enough to surround the inlets of the chips when placed above.
For the lysis reservoir, depending on the device size used, cut out the center to barely surround the barcoded region of the tissue (1x1mm for the 10µm channel device, 2.5x2.5mm for the 25µm channel device).

NOTE: The reservoirs are reusable. Thoroughly wash with 70% ethanol after each use.

CRITICAL: Be careful when cutting out the Chips A and B. Make sure not to cut across any patterned areas of the silicon wafer mold, otherwise it will need to be replaced and re-fabricated.
Preparing Barcodes
1h
Thoroughly mix the ligation linker with each barcodes A1-50 B1-50 at a 1:1 ratio.
Place the 50 mixes in a thermal cycle and heat to 97˚C to anneal.
Slowly cool to room temperature at a rate of -0.1˚C/sec.
Store at -20˚C for up to 6 months.
Preparing the Tissue Slide
1d
FFPE tissue sections were cut into sections with a thickness < 10 um and were placed at the center of a poly-L-lysine glass slide. Sections were stored at -80 ˚C until use.
Tissue sections were taken out from the freezer and allowed to come up to room temperature for 10 min until all the moisture disappears.              
Tissue Preparation
1h
Bake slides at 60 degrees Celsius for 2 hours.
Perform deparaffinization as follows:
*  100% Xylene 5 minutes
*  100% Xylene 5 minutes
*  100% Ethanol for 5 minutes
*  90% Ethanol for 5 minutes
*  70% Ethanol for 5 minutes
*  50% Ethanol for 5 minutes
* Deionized water for 5 minutes
5. Antigen-retrieval using 1X Tris-EDTA pH 9
* Allow to boil
* Incubate slide for ~ 10 minutes
* Allow to cool to room temperature (Can incorporate cooling on ice to speed up the process)
Permeabilization: 500μL 0.5% Triton X-100 in PBS was added on top of the tissue, and tissues were permeabilized for 20 min at room temperature.
To stop permeabilization, 0.5X PBS-RI was used to clean up the tissue sections.
Take a full pre-scan image of the section at the desired optical resolution

Recommended 10X resolution.

In situ polyadenylation
Reagents
Poly(A) wash buffer: 20μL 5X Poly(A) Polymerase Reaction Buffer, 2μL RNase Inhibitor, 78μl nuclease-free water.

ABCD
Reagent nameConcentrationFor 1 chipFor 2 chips
Nuclease-free Water47.6μL95.3μL
5X Poly(A) Reaction Buffer5X15μL30μL
Poly(A) Polymerase600U/μL3μL6μL
ATP100mM0.38μL0.75μL
SuperaseIn RNase Inhibitor20U/μL6μL12μL
RNase Inhibitor40U/μL3μL6μL
Table 1: Poly(A) mix preparation

The tissue section was equilibrated by adding 100μL of Poly(A) wash buffer, incubated for 5 min at room temperature, followed by air drying.
Next, attach the PDMS reservoir, add 75μL of Poly(A) Mix to the reaction chamber, and seal with parafilm. Incubate at 37˚C for 25 min, in a wet box.
After incubation, 0.5X PBS-RI was used to clean up the tissue sections.
Bulk Tissue Reverse Transcription
1h
Dip in a 50 mL falcon tube with DI water and gently dry with air flow.
Primer sequences: 15T Biotin RT Primer: /5Phos/CATCGGCGTACGACTNNNNNNNNNN/iBiodT/TTTTTTTTTTTTTTTVN
Reverse transcription mix

ABCD
Reagent nameConcentrationFor 1 chipFor 2 chips
0.5X PBS-RI20μL40μL
5X Maxima RT buffer5X12μL24μL
30T Biotin RT Primer25uM10μL20μL
Maxima H Minus RT200U/μL6μL12μL
Nuclease-free Water8μL16μL
dNTPs (10mM)10mM3μL6μL
SuperaseIn RNase Inhibitor20U/μL0.8μL1.6μL
RNase Inhibitor40U/μL0.4μL0.8μL
Table 2: Reverse Transcription (RT) mix preparation
1X NEB buffer 3.1: 100μL 10X NEB buffer + 900μL Nuclease-free Water.
The 60μL RT Mix was loaded on the sample with PDMS reservoir and sealed with parafilm. Then, incubated at room temperature for 30 min, and 42˚C for 90 min, in a wet box.
After RT reaction, samples were flow washed with 1 mL Nuclease-free Water for 5 times, then incubated in 500μL 1X NEB buffer 3.1 for 5 min at room temperature, followed by air drying.
Ligation of Barcodes
Reagents
Ligation mix

ABCD
Reagent nameConcentrationFor 1 chipFor 2 chips
1X NEB buffer 3.11X100μL200μL
10X T4 Ligase buffer10X26μL52μL
Nuclease-free Water61.3μL122.6μL
T4 DNA Ligase400U/μL15μL30μL
5% Triton-X1005μL10μL
RNase Inhibitor40U/μL2μL4μL
SuperaseIn RNase Inhibitor20U/μL0.7μL1.4μL
Table 3: Preparation of ligation mix


Then, in a 96-well plate, add 4μL of the above Ligation Mix for each well.
For each well, 1μL of 25uM of prepared Barcode A with Linker1 (A1-A50) was added and mixed with the Ligation Mix.

Prepare the wash buffer as follows:
Wash buffer: 2mL PBS+20μL 10% Triton X-100+5μL SuperaseIn RNase Inhibitor.
Attaching PDMS chips and loading of ligation mix
1h
Attach Chip-A as shown in Figure 5 centered on the tissue and take another scan of the channels laying on the tissue (Figure 4C).

Figure 4. Assembly to create the setup for the PDMS reservoir and acrylic clamps. (A) Chip-A with the inlet reservoir and barcoding clamp. (B) Sideview of Chip-A. (C) The lysis reservoir with the lysis clamp on top of the barcoded tissue. (D) Sideview of the lysis setup.
Figure 5. Example Scanning of the Tissue Slide.
(C) Scan of Chip-A channels placed on the tissue.

Use the acrylic clamp to firmly hold the PDMS chip against the tissue.
Place the inlet reservoir above the inlets. The final mixture (5μL in total) was injected into each of the 50 inlets of the 1st PDMS and vacuumed through the channels, making sure all the channels were filled.
Attach the vacuum head to the outlets as shown in Figure 6 and pull the solution through the 50 channels.

NOTE: Make sure all channels are filled with solution.

Figure 6. Chip-A with Acrylic Clamp and House Vacuum attached.

Stop the vacuum and incubate at 37 ˚C for 15 mins. Take out the chips and check if there are missed channels induced by bubbles. Vacuum again if needed and further incubate for 30 mins.
After ligation reaction, aspirate remaining barcode A mixture from inlets/outlets.
Clean the channels by flushing 1mL Wash buffer for 5 min and peel off PDMS and flow wash the chip once with 1 mL Nuclease-free Water, followed by air dry.
In-Tissue Ligation of barcode B
1h
Attach Chip-B centered on the tissue.
Scan the channels over the tissue to ensure proper placement.
Figure 7: Example scanning of the tissue slide.
(D) Scan of Chip-B channels across the tissue.

Firmly clamp as before.
Prepare the ligation mixture.
Prepare barcode B mixture.
In a 96-well PCR plate, add 4 µL of the ligation mixture in each of 50 wells.
Add 1 µL of each 25 µM Barcode B1-50 to the wells and mix thoroughly.
Introduce final barcode B mixture to the tissue.
Pipet each barcode B mixture directly into the inlets.
Apply the vacuum and pull the solution until all channels are full. May take as short as 10 sec or as long as 2 min.

CRITICAL: Do not over vacuum! Make sure the inlets do not run out of barcode B mixture which will cause the channels to lose the mixture and fill with air.
Incubate in the humidified chamber at 37˚C for 30 min for in-cell ligation of barcode B (Figure 8).
Clean the channels with wash buffer for 10 min.
Prepare wash buffer by mixing 4 mL of 1X PBS, 40 µL of 10% Triton X-100, and 10 µL of SUPERase• InTM RNase Inhibitor.
Peel off Chip-B and dip the slide into a 50mL falcon tube with DI water and gently dry with air flow.
Take a final post-scan of the tissue section.
Figure 8. Example Scanning of the Tissue Slide.
(B) Final scan after barcoding and removing the device.

Lysis and Sub-library Generation
2h 30m
Place the lysis reservoir directly around the barcoded tissue region.

NOTE: Try to cover as little extra tissue as possible.
Tightly apply the lysis clamp.
Add lysis solution to lysis reservoir depending on channel dimensions (~20 µL for the 25 µm channels, ~10 µL for the 10 µm channels).
Lysis solution is made with 50 µL 1X PBS, 50 µL of 2X lysis buffer, and 10 µL of proteinase K solution (20mg/mL).
2X lysis buffer is made up of 20 mM Tris (pH 8.0), 400 mM NaCl, 100 mM EDTA (pH 8.0), and 4.4% SDS.
Place in the humidified chamber and tightly wrap the chamber with parafilm and incubate at 55˚C for 2 hours.
Collect the lysate into a 1.5 mL centrifuge tube. Add the same amount of extra lysis solution to wash out the reservoir and retrieve as much lysate as possible.
Immediately store at -80˚C until next step.

Pause Point: Stop here for the first day. The lysate should be stable at -80˚C for up to 2 weeks. The Methods Video S1 shows up to this step.
Video

cDNA Purification
1h 30m
The cDNA is purified and bound to Dynabeads™ MyOne™ Streptavidin C1 beads in this step.

Prepare 40 µL DynabeadsTM MyOneTM Streptavidin C1 beads (Thermofisher) per sub-library.
Wash 3 times with 800 µL of 1X B&W buffer with 0.05% Tween-20.
Resuspend beads in 100 µL 2X B&W buffer + 2 µL SUPERase• InTM RNase Inhibitor.
Purify lysate following the DNA Clean & Concentrator with Zymo-spin IC Columns (RPI Research Products) protocol. Use 100 µL water to elute DNA.
Add 100 µL of resuspended DynabeadsTM MyOneTM Streptavidin C1 magnetic beads to each lysate.
Rotate at 30 RPM and room temperature for 60 min for binding to occur.
Wash beads.
Wash twice with 400 µL of 1X B&W buffer with 5 min rotation after resuspending beads.
Wash once more with 400 µL 10 mM Tris containing 0.1% Tween-20 with 5 min rotation after resuspension.
Template Switch
2h
This step performs template switching and adds the second PCR handle.

Resuspend the streptavidin beads bound with cDNA in solution containing 44 µL of 5X Maxima RT buffer (ThermoFisher), 44 µL of 20% Ficoll PM-400 solution, 22 µL of 10 mM dNTPs each (ThermoFisher), 5.5 µL RNase Inhibitor (Enzymatics), 11 µL of Maxima H Minus Reverse Transcriptase (ThermoFisher), 5.5 µL of 100 µM of template switch primer, and 88 µL of RNase-free water.
Rotate beads at 30 RPM and room temperature for 30 min followed by rotation at 42˚C for 90 min.
PCR
2h
Wash beads and resuspend.
Wash once with 400 µL of 10 mM Tris and 0.1% Tween-20 solution and once more with 400 µL of RNase-free water.
Resuspend in the PCR mix solution containing 110 µL of 2X Kapa HiFi HotStart Master Mix (Kapa Biosystems), 8.8 µL each of 10 µM stocks of primers 1 and 2, and 92.4 µL of RNase-free water. Transfer 200 µL to four PCR tubes with 50 µL in each.
Perform PCR to detach cDNA from beads.
StepsTemperatureTimeCycles
Initial Denaturation 95˚C 3 min 1
Denaturation 98˚C 20 sec 5
Annealing 65˚C 45 sec
Extension 72˚C 3 min
PCR Cycling Conditions

Remove the DynabeadsTM from the PCR solution using a magnetic tube holder.
Add Evagreen (Biotium) at a 1X concentration.

NOTE: This step is optional.
Perform PCR again with the following thermocycling conditions. Cycling can also be halted once the qPCR signal begins to plateau.
StepsTemperatureTimeCycles
Initial Denaturation 95˚C 3 min 1
Denaturation 98˚C 20 sec 15 cycles
Annealing 65˚C 20 sec
Extension 72˚C 3 min
Final Extension 72˚C 5 min 1
Hold 4˚C forever
Removal of ribosomal RNA
rRNA removal using Bio-Rad kit. Link: SEQuoia RiboDepletion Kit (17006487)
After rRNA removal, another 10 cycle PCR using 200uL PCR reaction system (100uL 2X Kapa HiFi) using direct ligation primer and Illumina N70X primer
cDNA Purification
40m
Add 80 µL KAPA pure beads for 100 µL of the cDNA sample.

NOTE: Ensure that beads have been equilibrated to room temperature and are fully resuspended before use.
Mix thoroughly by pipetting.
Incubate the tubes at room temperature for at least 10 min to bind the cDNA to the beads.
Place the tubes on a magnet to capture the beads and incubate until the liquid is clear.
Carefully remove and discard the supernatant.
Keep the tubes on the magnet and add 200 µL of 80% ethanol.
Incubate the tubes at room temperature for at least 30 sec.
Carefully remove and discard the ethanol.
Repeat steps 86-88.
Try to remove all residual ethanol and dry the beads at room temperature for 3-5 min or until all the ethanol has evaporated.

CRITICAL: Over-drying may result in reduced yield.
Remove the tubes from the magnet and resuspend the beads in 15 µL of PCR-grade water or elution buffer depending on downstream application.
Incubate the tubes at room temperature for at least 10 min to elute the cDNA off the beads.
Place the tubes back on the magnet and incubate until the liquid is fully clear.
Transfer the clear supernatant to a new tube.
Perform Bioanalyzer QC to obtain cDNA length profile and concentration.

Pause Point: Stop here for the second day. Store at 4˚C for 1-2 weeks or up to 6 months at -20˚C.
Library Preparation
1h 30m
PCR product from previous step can be sequenced.