For the 5 GATC-overhang two-oligo adaptors set up the following reactions in 96-well plate format:
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1 Supplementary Protocol 1. Adaptor preparation: For the 5 GATC-overhang two-oligo adaptors set up the following reactions in 96-well plate format: Per reaction X96 10X NEBuffer 2 10 µl 10 µl x GATC Biotin Forward oligo (100 um) 45 µl ~ Indexed Reverse oligo (100 um) 45 µl ~ For the indexed Y-adaptors, set up the following reactions in 96-well plate format: Per reaction X96 10X NEBuffer 2 10 µl 10 µl x 96 Barcoded Y-adaptor HI (100 um) 20 µl ~ Barcoded Y-adaptor LO (100 um) 20 µl ~ Water 50 ul 50 ul x 96 For both adaptors, heat to 98 o C for 6 min, then ramp down (-0.1 o C / s or slower) to RT. The final concentration of the 5 -GATC internal bridge adaptors is 45 um. The final concentration of Y- adaptors is 20 um Check the annealing efficiency by running oligos on 4% DNA Agarose gel 2. Cell lysis and chromatin digestion with DpnII (10 million cells total) 2.1) Thaw a vial of 2% Formaldehyde-fixed human cells (HeLa S3 / HAP1 / GM12878 / K562) and a vial of 2.5% fixed Patski and 2% fixed MEFs at 37 o C for sec. 2.2) Resuspend in 1 ml of ice-cold cell lysis buffer containing protease inhibitor cocktail (Roche). 2.3) Transfer 2.5E6 of each type of cell to separate tubes. 2.4) Centrifuge for 30 seconds at 800xg at RT. 2.5) Discard the supernatant and resuspend the pellet in each tube in 95 µl of water and 5 µl 10% SDS. 2.6) Incubate at 60 o C for 10 min. 2.7) Add 100 µl of water and 25 µl 10% Triton X-100 to each tube and mix well. 2.8) Incubate at 37 o C for 10 min. 2.9) Add 40 µl NEBuffer 3.1, 8 µl DpnII (10 U/ul) and 120 µl water to each tube, mix well. 2.10) Incubate at 37 o C overnight. 2.11) Centrifuge for 60 seconds at 850xg at RT.
2 2.12) Discard the supernatant and resuspend the pellet in 384 µl water and 6 µl 100X BSA 1 in each tube, mix well. 3. Ligation of the biotin-labeled, barcoded Bridge adaptors (1E5 cells / well, 10 µl reaction volume in at least 1 96 well plate) 3.1) Distribute T4 DNA ligase/buffer to each well of one (or more) 96 well plates (below master mix volumes are for 2 plates, with 3 µl per well), s Per reaction Water 380 (1.4x220) µl 50% PEG (0.5x220) µl 10X T4 DNA Ligase Buffer 220 (1x220) µl T4 DNA ligase (5 U/ µl) 24 (0.15x220) µl 3.2) Distribute 2 ul of biotinylated barcoded double-stranded bridge adaptor (45 µm) to each well of each 96-well plate using a multi-channel pipette. 3.3) Distribute 5 µl nuclei to each well. 3.4) Incubate at 16 o C overnight. 3.5) Distribute 1 µl 0.25 M EDTA to each well to inactivate ligation reaction. 3.6) On ice, pool the samples from each well together (1 ml) into two 1.5ml tubes. 3.7) Centrifuge at 850xg at RT for 90 seconds. 3.8) Resuspend in 291 µl of water, 6 µl 10%SDS, and 3 µl of 100X BSA, and combine the four tubes. 3.9) Centrifuge at 850xg at RT for 40 seconds. 3.10) Resuspend in 294 µl of water, 3 µl 10%SDS, and 3 µl of 100X BSA. 3.11) Incubate at 37 o C for 3 min. 3.12) Centrifuge at 850xg at RT for 40 seconds. 3.13) Resuspend in 294 µl of water, 3 µl 10%SDS, and 3 µl of 100X BSA. 3.14) Centrifuge at 850xg at RT for 40 seconds. 3.15) Resuspend in 237 µl of water, and 3 µl of 100X BSA, mix well. 3.16) Add 5 µl 10% Triton X-100, mix well. 4. Phosphorylation 4.1) Prepare the PNK-ligation master-mix for the nuclei as follows: 1 Stock conc. = 10 mg / ml
3 4.2) Incubate both at 37 o C for 1 hr. 5. In situ ligation 5.1) Add the following to the above tube: Nuclei 250 µl 10x T4 DNA Ligase buffer 30 µl PNK (10 U/ µl) 20 µl Water 615 µl 10x T4 DNA Ligase buffer 70 µl T4 DNA ligase (5 U/ µl) 8 µl 100X BSA 7 µl 5.2) Mix well, and incubate at RT for 4 hr. Divide into two tubes. 5.3) Centrifuge at 850xg at RT for 90 seconds. 5.4) Resuspend in 294 µl of water, 3 µl 10%SDS, and 3 µl of 100x BSA. 5.5) Incubate at 37 o C for 3 min. 5.6) Centrifuge at 850xg at RT for 40 seconds. 5.7) Resuspend in 294 µl of water, 3 µl 10%SDS, and 3 µl of 100x BSA. 5.8) Centrifuge at 850xg at RT for 40 seconds. 5.9) Resuspend in 294 µl of water, 3 µl 10%SDS, and 3 µl of 100x BSA. 6. Serial dilution and reverse cross-linking 6.1) Carry out serial dilution as follows: transfer 5 µl from 5.9) to 980 µl water plus 10 µl 100X BSA and 5 µl 10% SDS, mix well; then transfer 100 µl of diluted nuclei to 885 µl water plus 10 µl 100X BSA and 5 µl 10% SDS and mix well; then transfer 100 µl of diluted nuclei to 885 µl water plus 10 µl 100X BSA and 5 µl 10% SDS, and mix well. There should be ~1 nucleus per 2 µl solution. At last distribute 20 µl per well to a Lo-bind 96- well PCR plate. 6.2) Make the following master mix for reverse crosslinking: 6.3) Distribute 20 µl to each well. Per plate (x110) Water 15.5 µl (1705) 10% SDS 0.5 µl (55) 2X B&W Buffer 2 µl (220) Proteinase K (20 mg / ml) 2 µl (220)
4 6.4) Incubate at 62 o C overnight. Plates can then be stored at -20 o C for later processing. At this point, 8-strip PCR tubes may be used for small-scale assays, and the remaining cells can be processed as a bulk control assay. 7. Binding to 10 ul of Ampure beads (160 µl) 7.1) Dilute 1200 µl Ampure beads with 7 ml AMPure buffer and mix well. 7.2) If the 96-well plate was stored at -20 o C, thaw at 65 o C for 10 min. 7.3) Add 40 µl water to each well. 7.4) Add 80 µl diluted AMPure beads to each well with 12-channel multipipette. Mix well. 7.5) Incubate at RT for 10 min. 7.6) Perform quick spin to bring volume to bottom of each well. 7.7) Place the plate in a 96-well magnet and discard the supernatant with multichannel pipette. 7.8) Wash two times with 160 µl 80% EtOH 7.9) Remove the residue solution. 7.10) Quick spin plate to get the beads to the bottom of each well. 7.11) Resuspend the beads in 5 µl water, mix well by moving the beads around on the magnet. 7.12) Incubate at 37 o C for 10 min. 7.13) Quick spin to get the beads to the bottom of each well cutter digestion (MseI + HaeIII) (10 µl) 8.1) Add 5 ul of the following reaction buffer to each well: Per plate (x110) Water 3.4 µl (374) 10X NEBuffer 2 1 µl (110) MseI (10 U / µl) 0.3 µl (33) HaeIII (10 U / µl) 0.3 µl (33) 8.2) Quickly spin plate to bring volume to bottom of each well. 8.3) Incubate at 37 o C for 20 h, 65 o C for 30 min, and 4 o C forever. 9. End-repair to remove dangling ends (15 µl) 9.1) On ice, add 5 ul of the following reaction mix to each well: Per plate (x110) Water 3.55 µl (391.5) 10X NEBuffer µl (55) 10 mm dntps 0.2 µl (22) Klenow (10 U / µl) 0.15 µl (16.5) Carrier plasmid DNA 2 (220 ng/ul) 0.6 µl (60) 2 We use puc19 as a carrier plasmid.
5 9.2) Quick spin plate. 9.3) Incubate at 18 o C for 10 min, 65 o C for 30 min., and 4 o C forever. 10. da-tailing (20 µl) 10.1) Add 5 ul of da-tailing reaction mix to each well: Per reaction (x110) 10X NEBuffer µl (55) 20 mm datp 1 µl (110) Klenow (exo - ) (5 U/ µl) 0.3 µl (33) Water 3.2 µl (352) 10.2) Quick spin. 10.3) Incubate at 37 o C for 45 min, 65 o C for 30 min., and 4 o C forever. 11. Ligation of sequencing adaptors (20 µl), 96 indexed adaptors 11.1) Add 19 ul ligation mix to each well: Per reaction (x110) Water 10.8 µl (1188) 10X T4 DNA Ligase Buffer 4 µl (440) 50% PEG µl (440) T4 DNA ligase (5 U/ µl) 0.2 µl (22) 11.2) Distribute 1 µl Indexed sequencing adaptor (20 µm) to each well. 11.3) Quick spin. 11.4) Incubate at 16 o C for 20 h, 70 o C for 10 min, and 4 o C forever 12. Purification and Biotin pull down 12.1) Combine each column of wells into ml low-binding tubes. 12.2) Use 100 µl EB to rinse each well and add to each respective tube. 12.3) Add 40 µl 3M NaOAc, 4 µl GlycoBlue, and 500 µl isopropanol to each tube, mix well. 12.4) Incubate tubes at -80 o C for 2 h. 12.5) Spin at max speed on a chilled tabletop centrifuge for 30 min. 12.6) Resuspend and combine all 12 pellets in 100 µl EB. 12.7) Use 100 µl EB to rinse each tube twice (final volume 300 ul EB). 12.8) Add 100 µl AMPure Beads and 200 µl AMPure buffer. 12.9) Incubate at RT for 5 min ) Wash two times with 80% EtOH ) Resuspend in 100 µl water in each tube ) Bind to 10 µl M-280 Beads in 200 µl 1X B&W buffer 12.13) Incubate at RT for 20 min ) Wash 4 times with 200 µl 1X B&W buffer containing 0.1% Tween ) Wash twice with 200 µl EB.
6 12.16) Resuspend in 30 µl EB in each tube. 13. Library amplification (80 µl) 13.1) Carry out 3 PCR reactions with the following reaction composition: 13.2) Carry out PCR amplification: Water 22 µl 2X KAPA Hi-Fi Master Mix 40 µl Illumina F (10 um) 4 µl Barcoded Illumina R (10 um) 4 µl Beads 10 µl 98 o C, 2 min; 4 cycles: 98 o C, 20s; 60 o C, 30s; 72 o C, 45 sec cycles: 98 o C, 20s; 65 o C, 20s; 72 o C, 40 sec 13.3) Pool together all samples from each PCR reaction. 13.4) Add 30 µl AMPure beads and a volume of AMPure buffer necessary to achieve a 1:1 volumetric ratio of AMPure buffer + beads to sample. 13.5) Incubate 5 min at RT, and wash once with 0.6 ml 80% EtOH. 13.6) Resuspend in 200 µl water, add 155 µl AMPure buffer, mix well. 13.7) Incubate 5 min at RT, wash twice with 0.8 ml 80% EtOH. 13.8) Repeat three times. 13.9) After final purification, elute DNA with 30 µl EB and measure concentration using a Qubit ) Proceed to sequencing on a HiSeq 2500 instrument (PE 2x250 bp reads). 14. Library validation by EcoRI digestion (optional) 14.1) Digest a small aliquot of the final combinatorial single cell Hi-C library with EcoRI to estimate the portion of molecules with valid ligated junctions Experimental Control 10X Cutsmart Buffer 1µl 1µl PCR Product 1-2 µl (20 ng) 1-2 µl (20 ng) EcoRI-HF 1 µl 0 µl Water to 10 µl to 10 µl 14.2) Incubate at 37 o C for 1 hr. 14.3) Run the entire volume of the reaction on a 6% TBE-PAGE gel. Digested libraries should demonstrate a marked shift, as properly ligated molecules will be cut with EcoRI.
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