single-cell Micro-C protocol

Prepare 1 x BSA in PBS for Washing.

• Add 50µL 10% BSA (MACS 130-091-376, final 1x) to 50mL 1x PBS.

Prepare 1% Paraformaldehyde (1mL for 1 million cells), recover to room temperature.

• 1mL 32% PFA (EMS 15714, final 1%)
• 31mL 1x PBS

Prepare 3 mM DSG (1mL for 1 million cells).

• Dissolve 50 mg DSG (Thermo Fisher 20593) with 511µL DMSO to a final concentration of 300 mM.
• Add 300µL 300 mM DSG to 29.7mL 1 x PBS to a final concentration of 3 mM, protected from light.

Harvest cells.

• Collect cell growth with appropriate density and high viability.
• Wash cells once with 1x PBS.

Fixation.

• Resuspend cell pellets in 1% PFA, and incubate at room temperature with rotation for 10 min.
• At the end of incubation, add 2 M Tris-HCl ph 7.5 to a final concentration of 0.75 M, and incubate at RT for 5 min.
• Centrifuge at 3000 x g for 5 min in a swing bucket centrifuge.

Remove the supernatant, then wash twice with 5mL ice-cold 1 x BSA in PBS.

Crosslink with DSG.

• Resuspend in 3 mM DSG, and incubate at RT for 45 min with rotation.
• At the end of incubation, add 2 M Tris-HCl ph 7.5 to a final concentration of 0.75 M, and incubate at RT for 5 min.
• Centrifuge at 3000 x g for 5 min in a swing bucket centrifuge.

Count cells and aliquot.

• Remove the supernatant, then wash twice with 5mL ice-cold 1 x BSA in PBS.
• Count cell number, then aliquot 1 million cells each to 1.5 mL tube.
• Store at -80°C (up to a year).

Prepare Micro-C Buffers

10mL Prepare Micro-C Buffer 1 (300µL)

• 50µL 5 M NaCl (Invitrogen AM9760G; final 50 mM)
• 50µL 1 M Tris pH 7.5 (Invitrogen 15567027; final 10 mM)
• 25µL 1 M MgCl2 (Invitrogen AM9530G; final 5 mM)
• 5µL 1 M CaCl2 (Sigma 21115; final 1 mM)
• 50µL 5% Digitonin (Sigma D141-100MG, final: 0.05%)
• 50µL 100 x protease inhibitor (Roche 4693159001) (final: 1 X)
• 4.77mL nuclease-free H₂O

Prepare Micro-C Buffer 2 (10mL)

• 100µL 5 M NaCl (Invitrogen AM9760G; final 50 mM)

• 100µL 1 M Tris pH 7.5 (Invitrogen 15567027; final 10 mM)

• 100µL 1 M MgCl2 (Invitrogen AM9530G; final 10 mM)

• 9.7mLnuclease-free H₂O

Prepare Micro-C Buffer 3 (10mL)

• 500µL 1 M Tris pH 7.5 (Invitrogen 15567027; final 50 mM)
• 100µL 1 M MgCl2 (Invitrogen AM9530G; final 10 mM)
• 9.4mL nuclease-free H₂O

Dilute MNase.

• Make MNase Dilution Buffer (10 mM Tris-HCl ph 7.5, 50 mM NaCl, 1 mM EDTA, 50% Glycerol).
• Aliquot 10,000 Unit MNase (NEB M0247S, 2,000 U/µL) to 500µLMNase Dilution Buffer to a final concentration of 20 U/µL.
• Store at -80°C for several month.

Thaw 5 tubes of cell pellets on ice.

Resuspend in 100µL Micro-C Buffer 1, incubate on ice for 20 min, and aspiration every 5 min.

Centrifuge at 800 x g at 4°C for 5 min.

Take 5 ul to check digestion efficiency, remaining centrifuge at 800 x g at 4°C for 5 min. Carefully discard the supernatant, and wash once with 500µLMicro-C Buffer 2. Store at -80°C (stable for up to several weeks) or proceed to End Repair.

Check Digestion Efficiency

• Take 5µL above sample to check digestion efficiency.
• Add 85 ul 1 x PBS, 5 ul 10% SDS (Sigma, 71736-100ML), and 5 ul 20 mg/mL ProteaseK (QIAGEN, 19131).
• incubate at 65°C for 2 hr.
• Purify with DNA clean column (ZYMO, D4014).
• Run capillary electrophoresis (Fragment Analyzer/TepeStation/Bioanalyzer) to analyze digestion efficiency.

  

Thaw the cell pellet (with the most appropriate fragment size) on ice.

Prepare 0.5% SDS.

• Add 25µL 10% SDS to 475µL Micro-C Buffer 2 to a final concentration of 0.5%.

Solubilize Chromatin.

• Resuspend the cell pellets in 50µL 0.5% SDS.
• Incubate at 62°C for 10 min.
• At the end of incubation, add 50µL 5% Triton X-100 to quench SDS. Incubate at 37°C for 15 min.

Centrifuge at 800 x g for 5 min, carefully remove supernatant.

Prepare End Repair Mix 1 (45µL each sample).

• 5µL 10 X NEBuffer 2.1 (NEB B7202S)
• 1µL100 mM ATP (Thermo R0441)
• 2.5µL 100 mM DTT (final: ~5 mM)
• 2.5µL 10 U/uL T4 PNK (NEB M0201S) (final: ~0.5 U/uL)
• 34µL nuclease-free H₂O

Resuspend in 45µL End Repair Mix1, and incubate at 37°C for 15 min.

At the end of incubation, add 5µL 5 U/uL Klenow Fragment (NEB M0210S), incubate at 37°C for another 15 min.

Prepare End Repair Mix2 (25µLeach sample)

• 2.5µL 10 X T4 DNA Ligase Buffer (NEB B0202S)
• 0.5µL 10 mM (each) dNTP (NEB N0447S)
• 0.125µL 20 mg/mL BSA (NEB B9000S)
• 21.875µL nuclease-free H₂O

After Klenow incubation, add 25µL End Repair Mix2 to each tube, incubate at 23°C for 45 min (shake 30 s and pause 2 min).

Add 5 ul 0.5 M EDTA (Invitrogen AM9260G).

Incubate at 65°C for 20 min.

Wash once with 1mL Micro-C Buffer 3.

Prepare Ligation Mix (250µL each sample).

• 25µL 10 X T4 DNA Ligase Buffer (NEB B0202S)
• 1.25µL 20 mg/mL BSA (NEB B9000S)
• 12.5µL 400 U/uL T4 DNA Ligase (NEB M0202S)
• 211.25µL nuclease-free H₂O

Resuspend the cell pellet in 250µL Ligation Mix.

Take 25µL as ligation efficiency control.

Check Ligation Efficiency

• Take 25µL ligation product above sample to check ligation efficiency.

• Add 65 ul 1 x PBS, 5 ul 10% SDS (Sigma, 71736-100ML), and 5 ul 20 mg/mL ProteaseK (QIAGEN, 19131).

• incubate at 65°C for 2 hr.

• Purify with DNA clean column (ZYMO, D4014).

• Run capillary electrophoresis (Fragment Analyzer/TepeStation/Bioanalyzer) to analyze ligation efficiency.

  

Primers

• META 16 sequence

A	B
META-16-1	GGCACCG AAAA
META-16-2	CTCGGCGA TAAA
META-16-3	GGTGGAGC ATAA
META-16-4	CGAGCGCA TTAA
META-16-5	AGCCCGGT TATA
META-16-6	TCGGCACC AATA
META-16-7	GCCTGTGG ATTA
META-16-8	GCGACCCT TTTA
META-16-9	GCATGCGG TAAT
META-16-10	GCGTTGCC ATAT
META-16-11	GGCCGCAT TTAT
META-16-12	ACCGCCTC TATT
META-16-13	CCGTGCCA AAAT
META-16-14	TCTCCGGG AATT
META-16-15	CCGCGCTT ATTT
META-16-16	CTGAGCTCG TTTT

• META Transposon | A | B | | --- | --- | | META Transposon | 5'-[META sequence]-AGATGTGTATAAGAGACAG-3' | | 19 bp ME | 5'-/phos/-CTGTCTCTTATACACATCT-3' |

• First Step PCR Primers

A	B
First PCR primers	5'-[META sequence]-AGATGTGTATAAG-3'

Here we use 12 x 8 barcode combinations to distinguish individual cells.

• META16-ADP1 cell barcode | A | B | | --- | --- | | 1-1 | GATATG | | 1-2 | ATACG | | 1-3 | CCGTCTG | | 1-4 | TGCG | | 1-5 | GAACTCG | | 1-6 | ATGTAG | | 1-7 | CCCG | | 1-8 | TATGT | | 1-9 | GAGTAAG | | 1-10 | ATCG | | 1-11 | CCTAG | | 1-12 | TGACCG |

• META16-ADP2 cell barcode

A	B
2-1	ACTCTA
2-2	AGAGCAT
2-3	GGTATG
2-4	TCGATGC
2-5	CTACTAG
2-6	TATGCA
2-7	CACACGA
2-8	GTCGAT

Second Step Barcoded PCR Primers

A	B
META16-ADP1	5'-CTTTCCCTACACGACGCTCTTCCGATCT-[cell barcode]-[META sequence]-AGATGTGTATAAG-3'
META16-ADP2	5'-GAGTTCAGACGTGTGCTCTTCCGATCT-[cell barcode]-[META sequence]-AGATGTGTATAAG-3'

Prepare Reagents.

Prepare 60 mg/mL Protease.

• Dissolve 1 vial QIAGEN protease (QIAGEN 19155) in 2.78 mL 50% glycerol.
• Aliquot and store at -80°C (stable for up half a year).

Cell Triton Lysis Buffer (1mL).

• 10µL 1 M Tris pH 8.0 (Invitrogen AM9855G, final 10 mM)
• 4µL 5 M NaCl (Invitrogen AM9760G , final 20 mM)
• 2µL 0.5 M EDTA (Invitrogen AM9260G, final 1 mM)
• 10µL 10% Triton X-100 (Sigma 93443, final 0.1%)
• 25µL 1 M DTT (Sigma 646563, final 25 mM)
• 5µL 100 µM Carrier ssDNA (final 100 nM)
• Add H₂O to 1 mL
• 25µL 60 mg/mL QIAGEN protease (QIAGEN 19155, final 1.5 mg/mL) (freshly add when used)

A	B
Carrier ssDNA	TCAGGTTTTCCTGAA

2 X Transposition Buffer (1mL)

• 20µL 1 M Tris-HCl pH 8.0 (Invitrogen AM9855G, final 20 mM)
• 10µL 1 M MgCl₂ (Thermo Fisher AM9530G, final 10 mM)
• 400 ul 40% (w/w) Polyethylene glycol 8000 (Sigma P1458, final 16% (w/w) )
• 570µLnuclease-free H₂O Store at -20℃ if needed

Pick single cells.

Use mouth pipette or Fluorescence-activated cell sorting (FACS) to pick single cell into single PCR tubes or 96-well plates containing 2µL cell lysis buffer.

Cell lysis.

Incubate as:

50℃, 1 hr

65℃, 1 hr

70℃, 15 min

Stored at -80℃ or proceed to transposon-based amplification.

Transposition (8µLfor each well, below recipe for 1 96-well plate).

1. Prepare Transposition Mix

• 440µL 2 X Transposition Buffer
• Add META transposome* to a final concentration of 0.3 nM
• Add nuclease-free H₂O to 880µL
• Transposome: META transposome is optional. Alternatively, Nextera transposome can be used (if using Nextera procedure, please refer to "Dip-C (Part 2: Whole-genome Amplification with Nextera)") on protocol.io.

2. Add 8µL Transposition Mix to each well with a multi-channel pipette, vortex to mix.

3. Incubate at 55℃ for 10 min.

Tn5 Stop.

• Prepare Tn5 Removal Mix (250 mM NaCl, 37.5 mM EDTA, 2 mg/mL QIAGEN protease).

• Add 2µL Tn5 Removal Mix to each tube, and mix thoroughly.

• Incubate as: 50℃, 30 min

  70℃, 15 min
  

Pre-amplification.

Prepare Preamplification Mix (16µL each well, below recipe for 1 96-well plate)

• 1540µL 2 X Q5 High-fidelity Master Mix (NEB M0492L)
• 98.6µL 50 µM First Step PCR Primers (final 0.1 µM each)
• 55µL 100 mM MgCl₂
• 66.4µL nuclease-free H₂O

Add 16µL Preamplification Mix each well with a multi-channel pipette, and vortex to mix.

Amplified as:

72℃, 5 min

98℃, 30 s

12 cycles of [98℃ for 10 s, 62℃ for 30 s, 72℃, 2 min]

65℃, 5 min.

Cell Barcoding PCR.

• Add 1µL individual 50 μM barcoded META16-ADP1 Primer and 1µL individual 50 μM indexed META16-ADP2 Primer Mix to each well with a multi-channel pipette.

• Incubate as: 98℃, 30 s

  3 cycles of [98℃ for 10 s, 62℃ for 30 s, 72℃, 2 min]

  65℃, 5 min

Purification.

• Pool a 96-well plate to purify.
• Purify with clean column (ZYMO, D4014).
• Quantify with Qubit Fluorometer.

Library preparation.

• Take 400 ng amplified product for each plate as input.

• Prepare Library Preparation Mix (50µL2 X Q5 High-fidelity Mater Mix, template (400 ng), 5µL 10 µM Indexed i5 primer, 5µL 10 µM Indexed i7 primer (Vazyeme N321/N322), nuclease-free H₂O to 100µL)

• Amplified as: 98℃, 30 s

  2 cycles of [98℃, 10 s, 68℃, 30 s, 72℃, 2 min] 
  
  72℃, 5 min
  

• Purify with PCR clean column (ZYMO D4014), then purify with 0.75 x AMPUre XP beads to select > 300 bp fragments for sequencing.

Sequencing.

META library only has 20% valid fragments having both P7 and P5 on either side, need to RUN qPCR quantify before loading.