Nov 18, 2020

Public workspaceDip-C (Part 2: Whole-genome Amplification with Nextera)

DOI
dx.doi.org/10.17504/protocols.io.bpt8mnrw
  • 1Stanford University
Longzhi Tan
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DOI: dx.doi.org/10.17504/protocols.io.bpt8mnrw
Protocol Citation: Longzhi Tan 2020. Dip-C (Part 2: Whole-genome Amplification with Nextera). protocols.io https://dx.doi.org/10.17504/protocols.io.bpt8mnrw
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: November 18, 2020
Last Modified: November 18, 2020
Protocol Integer ID: 44640
Protocol materials
ReagentTE, pH 8.0, RNase-freeThermo FisherCatalog #AM9849
ReagentQIAGEN Protease (7.5 AU)QiagenCatalog #19155
ReagentNaCl (5 M), RNase-freeThermo FisherCatalog #AM9760G
ReagentEDTA (0.5 M), pH 8.0, RNase-freeThermo FisherCatalog #AM9260G
Reagent1M DL-Dithiothreitol solution (DTT)Merck MilliporeSigma (Sigma-Aldrich)Catalog #646563
ReagentTriton X-100, 10% solution Merck MilliporeSigma (Sigma-Aldrich)Catalog #93443
ReagentTris (1 M), pH 8.0, RNase-freeThermo FisherCatalog #AM9855G
ReagentTAPS Buffer (1 M pH 8.5)Boston BioproductsCatalog #BB-2375
Reagent1M MgCl2Invitrogen - Thermo FisherCatalog #AM9530G
Reagent50% w/v Polyethylene glycol 8000Hampton ResearchCatalog #HR2-535
Reagent96 well LoBind PCR plates Semi-skirtedEppendorfCatalog #0030129504
ReagentFilm Sealing Roller for PCR PlatesBio-Rad LaboratoriesCatalog #MSR0001
ReagentAdhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001
ReagentDNA LoBind Tube 1.5ml EppendorfCatalog #022431021
ReagentHela Genomic DNA - 15 ugNew England BiolabsCatalog #N4006S
ReagentDNA LoBind Tube 1.5ml EppendorfCatalog #022431021
ReagentTriton X-100, 10% solution Merck MilliporeSigma (Sigma-Aldrich)Catalog #93443
ReagentNaCl (5 M), RNase-freeThermo FisherCatalog #AM9760G
ReagentEDTA (0.5 M), pH 8.0, RNase-freeThermo FisherCatalog #AM9260G
ReagentTTE Mix V50VazymeCatalog #TD501
ReagentBSA, molecular biology grade, 20 mg/ml New England BiolabsCatalog # B9000S
ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L
ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L
ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L
ReagentDeoxynucleotide Solution Mix - 8 umol of eachNew England BiolabsCatalog #N0447S
Reagent1M MgCl2Invitrogen - Thermo FisherCatalog #AM9530G
ReagentTE, pH 8.0, RNase-freeThermo FisherCatalog #AM9849
ReagentDNA Clean & Concentrator-5 (Capped) 50 PrepsZymo ResearchCatalog #D4013
ReagentDNA Binding Buffer 100 mLZymo ResearchCatalog #D4004-1-L
ReagentTE, pH 8.0, RNase-freeThermo FisherCatalog #AM9849
ReagentQubit™ 1X dsDNA HS Assay KitThermo FisherCatalog #Q33231
ReagentBioAnalyzer High Sensitivity Chip Agilent TechnologiesCatalog #5067-4626
ReagentSPRIselect 60 mLBeckman CoulterCatalog #B23318
ReagentTE, pH 8.0, RNase-freeThermo FisherCatalog #AM9849
ReagentQubit™ 1X dsDNA HS Assay KitThermo FisherCatalog #Q33231
ReagentBioAnalyzer High Sensitivity Chip Agilent TechnologiesCatalog #5067-4626
Oligos
Oligos
Carrier ssDNA (same as in LIANTI and META):
  • TCAGGTTTTCCTGAA
  • Purification: standard desalting
  • Dissolve in 0.1 X TE (made from ReagentTE, pH 8.0, RNase-freeThermo FisherCatalog #AM9849 ) to a final concentration of Concentration100 micromolar (µM) .
  • Store at Temperature-20 °C .
Nextera i7 Index Primers:
  • 701: CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG
  • 702: CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGG
  • 703: CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGG
  • 704: CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGG
  • 705: CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGG
  • 706: CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGG
  • 707: CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGG
  • 708: CAAGCAGAAGACGGCATACGAGATCCTCTCTGGTCTCGTGGGCTCGG
  • 709: CAAGCAGAAGACGGCATACGAGATAGCGTAGCGTCTCGTGGGCTCGG
  • 710: CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGG
  • 711: CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGG
  • 712: CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGG
  • and the following if > 96 cells need to be sequenced at the same time (e.g. NovaSeq):
  • 714: CAAGCAGAAGACGGCATACGAGATTCATGAGCGTCTCGTGGGCTCGG
  • 715: CAAGCAGAAGACGGCATACGAGATCCTGAGATGTCTCGTGGGCTCGG
  • 716: CAAGCAGAAGACGGCATACGAGATTAGCGAGTGTCTCGTGGGCTCGG
  • 718: CAAGCAGAAGACGGCATACGAGATGTAGCTCCGTCTCGTGGGCTCGG
  • 719: CAAGCAGAAGACGGCATACGAGATTACTACGCGTCTCGTGGGCTCGG
  • 720: CAAGCAGAAGACGGCATACGAGATAGGCTCCGGTCTCGTGGGCTCGG
  • 721: CAAGCAGAAGACGGCATACGAGATGCAGCGTAGTCTCGTGGGCTCGG
  • 722: CAAGCAGAAGACGGCATACGAGATCTGCGCATGTCTCGTGGGCTCGG
  • 723: CAAGCAGAAGACGGCATACGAGATGAGCGCTAGTCTCGTGGGCTCGG
  • 724: CAAGCAGAAGACGGCATACGAGATCGCTCAGTGTCTCGTGGGCTCGG
  • 726: CAAGCAGAAGACGGCATACGAGATGTCTTAGGGTCTCGTGGGCTCGG
  • 727: CAAGCAGAAGACGGCATACGAGATACTGATCGGTCTCGTGGGCTCGG
  • Purification: standard desalting
  • Dissolve in 0.1 X TE to a final concentration of Concentration50 micromolar (µM) .
  • Dilute with 0.1 X TE to Concentration12.5 micromolar (µM) in PCR tubes.
Nextera i5 Index Primers:
  • 501: AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTC
  • 502: AATGATACGGCGACCACCGAGATCTACACCTCTCTATTCGTCGGCAGCGTC
  • 503: AATGATACGGCGACCACCGAGATCTACACTATCCTCTTCGTCGGCAGCGTC
  • 504: AATGATACGGCGACCACCGAGATCTACACAGAGTAGATCGTCGGCAGCGTC
  • 505: AATGATACGGCGACCACCGAGATCTACACGTAAGGAGTCGTCGGCAGCGTC
  • 506: AATGATACGGCGACCACCGAGATCTACACACTGCATATCGTCGGCAGCGTC
  • 507: AATGATACGGCGACCACCGAGATCTACACAAGGAGTATCGTCGGCAGCGTC
  • 508: AATGATACGGCGACCACCGAGATCTACACCTAAGCCTTCGTCGGCAGCGTC
  • and the following if > 96 cells need to be sequenced at the same time (e.g. NovaSeq):
  • 510: AATGATACGGCGACCACCGAGATCTACACCGTCTAATTCGTCGGCAGCGTC
  • 511: AATGATACGGCGACCACCGAGATCTACACTCTCTCCGTCGTCGGCAGCGTC
  • 513: AATGATACGGCGACCACCGAGATCTACACTCGACTAGTCGTCGGCAGCGTC
  • 515: AATGATACGGCGACCACCGAGATCTACACTTCTAGCTTCGTCGGCAGCGTC
  • 516: AATGATACGGCGACCACCGAGATCTACACCCTAGAGTTCGTCGGCAGCGTC
  • 517: AATGATACGGCGACCACCGAGATCTACACGCGTAAGATCGTCGGCAGCGTC
  • 518: AATGATACGGCGACCACCGAGATCTACACCTATTAAGTCGTCGGCAGCGTC
  • 520: AATGATACGGCGACCACCGAGATCTACACAAGGCTATTCGTCGGCAGCGTC
  • Purification: standard desalting
  • Dissolve in 0.1 X TE to a final concentration of Concentration50 micromolar (µM) .
  • Dilute with 0.1 X TE to Concentration12.5 micromolar (µM) in PCR tubes.
Reagents
Reagents
Prepare 60 mg/mL Qiagen Protease:
  • Add Amount2.78 mL water to one vial of ReagentQIAGEN Protease (7.5 AU)QiagenCatalog #19155 .
  • Vortex to mix.
  • Filter to sterilize.
  • Store at Temperature4 °C .
Prepare Lysis Buffer (Amount2 µL per cell; recipe below for Amount1 mL , or 4 96-well plates):
  • Amount20 µL ReagentTris (1 M), pH 8.0, RNase-freeThermo FisherCatalog #AM9855G (final: Concentration20 millimolar (mM) )
  • Amount4 µL ReagentNaCl (5 M), RNase-freeThermo FisherCatalog #AM9760G (final: Concentration20 millimolar (mM) )
  • Amount15 µL ReagentTriton X-100, 10% solution Merck MilliporeSigma (Sigma-Aldrich)Catalog #93443 (final: Concentration0.15 % (v/v) )
  • Amount25 µL Reagent1M DL-Dithiothreitol solution (DTT)Merck MilliporeSigma (Sigma-Aldrich)Catalog #646563 (aliquoted and stored at Temperature-20 °C ; final: Concentration25 millimolar (mM) )
  • Amount2 µL ReagentEDTA (0.5 M), pH 8.0, RNase-freeThermo FisherCatalog #AM9260G (final: Concentration1 millimolar (mM) )
  • Amount5 µL 100 uM Carrier ssDNA (final: Concentration500 nanomolar (nM) )
  • Amount929 µL water
  • Vortex to mix.
  • Store at Temperature-20 °C if needed.
Prepare Transposition Buffer (Amount8 µL per cell; recipe below for Amount1 mL , or 1 96-well plate):
  • Amount12.5 µL ReagentTAPS Buffer (1 M pH 8.5)Boston BioproductsCatalog #BB-2375 (final: Concentration12.5 millimolar (mM) )
  • Amount6.25 µL Reagent1M MgCl2Invitrogen - Thermo FisherCatalog #AM9530G (final: Concentration6.25 millimolar (mM) )
  • Amount200 µL Reagent50% w/v Polyethylene glycol 8000Hampton ResearchCatalog #HR2-535 (final: Concentration10 Mass / % volume )
  • Amount781.25 µL water
  • Vortex to mix.
  • Store at Temperature-20 °C if needed.
Prepare Transposome Removal Buffer (Amount2 µL per cell; recipe below for Amount1 mL , or 4 96-well plates):
  • Amount60 µL ReagentNaCl (5 M), RNase-freeThermo FisherCatalog #AM9760G (final: Concentration300 millimolar (mM) )
  • Amount90 µL ReagentEDTA (0.5 M), pH 8.0, RNase-freeThermo FisherCatalog #AM9260G (final: Concentration45 millimolar (mM) )
  • Amount1 µL ReagentTriton X-100, 10% solution Merck MilliporeSigma (Sigma-Aldrich)Catalog #93443 (final: Concentration0.01 % (v/v) ; for ease of pipetting)
  • Amount849 µL water
  • Vortex to mix.
  • Store at Temperature-20 °C if needed.
Lysis (Skip if Performing Positive Control)
Lysis (Skip if Performing Positive Control)
2h 30m
2h 30m
Prepare Dip-C Lysis Buffer (Amount2 µL per cell; recipe below for 4 96-well plate + 25%):
  • Amount960 µL Lysis Buffer
  • Amount0.24 µL 60 mg/mL Qiagen Protease (final: Concentration15 μg/mL )
  • Vortex to mix.
  • Aliquot to Amount78 µL in 12-strip tubes.
Add Amount2 µL Dip-C Lysis Buffer per well to a Reagent96 well LoBind PCR plates Semi-skirtedEppendorfCatalog #0030129504 .
Seal the plate with ReagentAdhesive PCR Plate SealBio-Rad LaboratoriesCatalog #MSB1001 and ReagentFilm Sealing Roller for PCR PlatesBio-Rad LaboratoriesCatalog #MSR0001 .
Centrifuge at Centrifigation1000 x g briefly.
Flow sort one cell per well (see Part 1 for details).
Centrifuge at Centrifigation1000 x g, 00:01:00 .
Lyse the cells by running (set lid temperature to Temperature75 °C to avoid evaporation; Amount2 µL volume; total: ~Duration01:15:00 ; minimize evaporation by sealing tight and closing the PCR machine lid tight):
  • Temperature50 °C for Duration01:00:00
  • Temperature70 °C for Duration00:15:00
  • Temperature4 °C forever
2h 30m
Store at Temperature-80 °C if needed (stable for a few months). For longer storage at Temperature-80 °C , sort cells into dry (empty) wells.

Positive Control (Optional)
Positive Control (Optional)
2h 30m
2h 30m
Prepare 100 pg/uL gDNA in a ReagentDNA LoBind Tube 1.5ml EppendorfCatalog #022431021 (recipe below for NEB HeLa gDNA; can be made from any human or mouse gDNA):
  • Amount1 mL water
  • Amount1 µL ReagentHela Genomic DNA - 15 ugNew England BiolabsCatalog #N4006S (final: Concentration100 pg/μL )
  • Vortex to mix.
Prepare Positive Control Solution in a ReagentDNA LoBind Tube 1.5ml EppendorfCatalog #022431021 (Amount2 µL per positive control; recipe below for Amount20 µL ):
  • Amount19 µL water
  • Amount1 µL 100 pg/uL gDNA (final: Concentration5 pg/μL )
  • Vortex to mix.
Add Amount2 µL Positive Control Solution per positive control well.
Transposition
Transposition
2h 30m
2h 30m
Make Transposition Mix (Amount8 µL per cell; recipe below for 96-well plate + 10%):
  • Amount844.8 µL Transposition Buffer
  • ~Amount1.6 µL 125 nM Homemade Nextera Transposome or ReagentTTE Mix V50VazymeCatalog #TD501
  • Pipette to mix.
  • Aliquot to Amount69 µL in 12-strip tubes.
Add Amount8 µL Transposition Mix per well, avoiding touching the liquid.
Vortex and spin down.
Transpose the genome by running (Amount10 µL volume; total: ~Duration00:10:00 ):
  • Temperature55 °C for Duration00:10:00
  • Temperature4 °C forever
20m
Stop
Stop
2h 30m
2h 30m
Prepare Stop Mix (Amount2 µL per cell; recipe below for 96-well plate + 25%):
  • Amount240 µL Transposome Removal Buffer
  • Amount0.4 µL 60 mg/mL Qiagen Protease (final: Concentration100 μg/mL )
  • Vortex to mix.
  • Aliquot to Amount19 µL in 12-strip tubes.
Add Amount2 µL Stop Mix per tube, avoiding touching the liquid.
Vortex and spin down.
Stop transposition by running (Amount12 µL volume; total: ~Duration01:00:00 ):
  • Temperature50 °C for Duration00:40:00
  • Temperature70 °C for Duration00:20:00
  • Temperature4 °C forever
2h
Amplification
Amplification
2h 30m
2h 30m
Make PCR Mix (~Amount11 µL per cell; recipe below for 96-well plate + 10%):
  • Amount528 µL Q5 Reaction Buffer (included with ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L )
  • Amount528 µL Q5 High GC Enhancer (included with ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L )
  • Amount63.36 µL ReagentDeoxynucleotide Solution Mix - 8 umol of eachNew England BiolabsCatalog #N0447S
  • Amount6.336 µL Reagent1M MgCl2Invitrogen - Thermo FisherCatalog #AM9530G
  • Amount26.4 µL ReagentBSA, molecular biology grade, 20 mg/ml New England BiolabsCatalog # B9000S
  • Amount26.4 µL ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L
  • Vortex to mix.
  • Aliquot to Amount97 µL in 12-strip tubes.
Add (per tube; avoid touching the liquid):
  • Amount11 µL PCR Mix
  • Amount1 µL 12.5 uM Nextera i5 Primer (final: Concentration500 nanomolar (nM) )
  • Amount1 µL 12.5 uM Nextera i7 Primer (final: Concentration500 nanomolar (nM) )
  • Arrange the indices so that no cells have the same index on each sequencing run.
Amplify by running (Amount25 µL volume; total: ~Duration01:00:00 ):
  • Temperature4 °C for Duration00:03:00 (to allow the lid to pre-heat)
  • Temperature72 °C for Duration00:03:00
  • Temperature98 °C for Duration00:00:20
  • 14 cycles of Temperature98 °C for Duration00:00:10 , Temperature62 °C for Duration00:01:00 , Temperature72 °C for Duration00:02:00
  • Temperature72 °C for Duration00:05:00
  • Temperature4 °C forever
1h 14m 30s
Store at Temperature-20 °C if needed.
Purification
Purification
2h 30m
2h 30m
Pool cells as desired and purify with ReagentDNA Clean & Concentrator-5 (Capped) 50 PrepsZymo ResearchCatalog #D4013 and Amount125 µL DNA Binding Buffer per cell (a 1:5 ratio; extra buffer can be purchased as ReagentDNA Binding Buffer 100 mLZymo ResearchCatalog #D4004-1-L ). Elute in Amount4 µL ReagentTE, pH 8.0, RNase-freeThermo FisherCatalog #AM9849 per cell.
For a 96-well plate, pooling can be done with a multi-channel pipette into a total of Amount12 mL Binding Buffer. Use 4-6 columns per plate and elute into a total of Amount400 µL ReagentTE, pH 8.0, RNase-freeThermo FisherCatalog #AM9849 .
Measure concentration with ReagentQubit™ 1X dsDNA HS Assay KitThermo FisherCatalog #Q33231 .
Measure size distribution with ReagentBioAnalyzer High Sensitivity Chip Agilent TechnologiesCatalog #5067-4626 .
Remove short fragments with 0.7 X ReagentSPRIselect 60 mLBeckman CoulterCatalog #B23318 . Elute into ReagentTE, pH 8.0, RNase-freeThermo FisherCatalog #AM9849 .

Measure concentration with ReagentQubit™ 1X dsDNA HS Assay KitThermo FisherCatalog #Q33231 .
Measure size distribution with ReagentBioAnalyzer High Sensitivity Chip Agilent TechnologiesCatalog #5067-4626 .