GSM9837101: SPT16_DMSO_Hi-C_B1_T2; Mus musculus; Hi-C
Cells were  resuspended in 1x DPBS w/o Ca2+ and Mg2+ to reach a solution of 1x10^6 cells/ml. The cell suspensions were then added 1% Formaldehyde and incubated at RT for 10 minutes on a rotator. The reaction was quenched using 0.25 M glycine and incubated at RT for 5 minutes. The samples were centrifuged at 1000g for 5 minutes, washed once with 1x PBS w/o Ca2+ and Mg2+ with 0.1% BSA and resuspended in 1x PBS w/o Ca2+ and Mg2+ with 0.1% BSA, reaching a solution of 4x10^6 cells/ml. EGS were dissolved in DMSO and 3 mM EGS were added to the cell suspension prior to incubation at RT for 40 minutes on a rotator. The reaction was quenched using 0.4 M glycine and incubated at RT for 5 minutes. The cells were centrifuged 1000g for 5 minutes and resuspended in 1x PBS w/o Ca2+ and Mg2+ reaching a concentration of 5x10^6 cells/ml. The crosslinked cells were aliquoted into Eppendorf tubes to generate samples either containing 5x10^6 or 1x10^6 cells. The aliquoted samples were centrifuged 2000g for 5 minutes and supernatant were carefully removed. Pelleted cells were snap frozen in liquid nitrogen and stored at -80°C until further processing. Hi-C 3.0 was performed as previously described157, with minor modifications. 5x10^6 double-crosslinked cells were lysed in lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal, 1x Roche cOmplete EDTA-free Protease Inhibitor) and digested for >16 hours with 10 U/µl Ddel and 50 U/µl DpnII, before incubating the samples for 4 hours with Biotin-fill-in-master mix (7 µl 10x NEBuffer 3.1, 1.5 µl 10 mM dCTP, 1.5 µl 10 mM dGTP, 1.5 µl 10 mM dTTP, 14 µl 1 mM biotin-dATP, 10 µl 5U/µl Klenow DNA polymerase, H2O). The proximal DNA fragments were ligated by adding 665 µl ligase master mix (120 µl 10x ligation buffer, 120 µl 10% Triton X-100, 12 µl 10 mg/ml BSA, 33.25 µl 400 U/µl T4 DNA ligase, H2O) to each sample and samples were incubated over night at 16°C with shaking. 50 µl 20 mg/ml proteinase K was added to each sample and incubated overnight at 65°C with shaking. For each sample, the DNA was purified with 2 volumes of Phenol-Chloroform-Isoamyl-Alcohol and the aqueous phase was transferred to clean tubes. The aqueous phase from each sample was then purified by ethanol precipitation and the solubilized pellet resuspended in 450 µl TLE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) prior to purification through a 10 kDa Amicon Ultra-0.5 centrifugal Filter Unit. The remaining samples were eluted in 80 ul TLE buffer and 10 µg DNA from each sample was used for further processing. Biotin-removal master mix (13 µl 10x NEBuffer 2.1, 3.25 µl 1 mM dATP, 3.25 µl 1 mM dGTP, 13 µl 300 U/ml T4 DNA polymerase, H2O) was added to each 10 µg DNA sample and incubated overnight according to the protocol. Sample were sonicated using a Covaris E220 according to manufacturer instructions to achieve fragment size distributions <500 bp followed by size selection using SPRI-Select beads to obtain Upper – and Lower fractions. Both fractions were eluted from the beads using 150 µl TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). To bind the DNA onto the streptavidin beads, 10 µl beads per sample were washed twice with 500 µl TBW buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween20) and the washed beads were resuspended in 150 µl per sample 2x B&W buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl). Each library sample were then added 150 µl of the beads-solution (1:1) and incubated with rotation at room temperature for 20 minutes. The beads now containing the DNA fragments were placed in a magnet and the cleared supernatant were removed. The beads were then washed twice in 300 µl TBW buffer and washed once in 100 µl 0.1% TE buffer before being resuspended in 50 µl 0.1% TE buffer and transferred to clean PCR-tubes. The samples were then processed using the NEBNext® UltraTM II DNA Library Prep Kit for Illumina® protocol step 1 and 2, following vendors instructions. Following end-prep and adapter ligation, the tubes were placed in a magnet and the supernatant was removed. The beads were washed once with 200 µl TBW and once with 100 µl 0.1% TE buffer. The beads were then resuspended in 20 µl 0.1% TE buffer and 1 µl from each sample were amplified in a 10 µl PCR reaction with 16 cycles to estimate the minimal number of cycles for the library PCR. Subsequently, the main samples were amplified in a 200 µl reaction with the appropriate number of cycles estimated from the 1 µl PCR test, and the amplified samples were purified with SPRI Select AMPure beads in the ratio 1:0.9 to deplete potential adapter dimers. The AMPure purified samples were eluted in 20 µl 0.1% TE buffer and were measured by Qubit. All samples were run on the TapeStation to estimate the average fragment size and diluted to 4 nM pools for paired-end sequencing. Samples were sequenced on the NextSeq2000 or NovaSeq.