title: GSM8125717: MicroC, control; Homo sapiens; Hi-C
library_construction_protocol: HCT116 cells were dissociated into single cells with TrypLE Express (Thermo Fischer Scientific) and washed with PBS once. The washed cells were crosslinked with 1% formaldehyde for 10 min and quenched with 125 mM glycine for 5 min. After washing with PBS, the cells were crosslinked with 3 mM DSG (Thermo Fisher) for 40 min and quenched with 400 mM glycine for 5 min. The crosslinked cells were washed with PBS and frozen in aliquots of 5 x 106 cells. Frozen cells were resuspended in Micro-C Buffer #1 (50 mM NaCl, 10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 1 mM CaCl2, 0.2% NP-40, 1x cOmplete EDTA-free (Roche)) and incubated on ice for 20 min. Cells were collected by centrifugation and resuspended with Micro-C Buffer #1 again. The treatment with MNase (NEB) at 37 C for 10 min fragmented chromatin. The reaction was stopped by EDTA and incubation at 65C for 10 min. The chromatin was collected by centrifugation and washed with Micro-C Buffer #2 (50 mM NaCl, 10 mM Tris-HCl pH 7.5, 10 mM MgCl2). To generate 5' overhangs, the ychromatin was resuspended with 50 μl end-repair mix (5 μl of 10 x NEBuffer 2.1, 2 μl of 100 mM ATP, 2.5 μl of 100 mM DTT, 8 μl of Large Klenow Fragment (NEB), 2 μl of T4 Polynucleotide Kinase (NEB).) and incubated at 37 C for 15 min. The DNA overhangs were filled with biotinylated nucleotides by addition of 100 μl Fill-in master mix (10 μl of 1 mM biotin-14-dATP (JBS), 10 μl of 1 mM biotin-11-dCTP (JBS), 1 μl of 10 mM dGTP (Thermo Fisher), 1 μl of 10 mM dTTP (Thermo Fisher), 10 μl of 10x NEBuffer 2.1). After incubation at 65 C for 20 min, the reaction was stopped by EDTA. The chromatin was collected with centrifugation and washed with 1x Ligase Reaction Buffer (NEB). For proximity ligation, the chromatin was resuspended with 500 μl Micro-C ligase mix (50 μl of 10x NEB Ligase buffer, 2.5 μl of 200x BSA (NEB), 5 μl of NEB T4 Ligase (NEB)) and incubated at RT form 2.5-3 h. For removal of biotin-dNTP from un-ligated ends, collected chromatin by centrifugation was resuspended with 100 μl of exonuclease mix (1x NEBuffer 1 (NEB) and 2 μl of 100 U/μl NEB Exonuclease III (NEB)) and incubate at 37C for 15 minutes. For deproteination and reverse crosslinking, 15 μl of Proteinase K (50 mg/mL) and 13 μl of 10% SDS (Invitrogen) were added and incubated at 55 C for 2h and at 65 C overnight. Ligated DNA was purified with QIAquick PCR purification kit (QIAGEN) and eluted in 110 μl of EB. Size selection of DNA was performed by AMPure XP beads (Beckman Coulter). 40 μl of beads were added to each reaction and incubated for 5 min. Using a magnetic stand, the clear solution was transferred to a fresh tube, and 20 μl of beads were added. After incubation for 5 min, the supernatant was removed, and beads were washed with 70 % ethanol without mixing. Beads were left to dry for 5 min and DNA was eluted with 50 μl of EB. For biotin pull-down, we used Dynabeads™ MyOne™ Streptavidin C1 beads. 5 μl beads were washed twice with 300 μl 1x Tween Washing Buffer (1x TWB; 5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl, 0.05% Tween 20) and suspended in 150 μl 2x Binding Buffer (2x BB; 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl). 100 μl H2O and 150 μl washed beads in 2x Binding Buffer were added to the sample and incubated rotation at RT for 20 min. Beads were washed with 1x TWB twice and with EB once. End repair and adaptor ligation was performed on beads using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) according to manufacturer's protocol. Then, beads were washed with 1x TWB twice and with EB once and resuspended with 15 μl of EB. DNA was amplified by PCR and DNA purification was performed using NEBNext Ultra II DNA Library Prep Kit, NEBNext® Multiplex Oligos for Illumina and AMPure XP beads following the manufacturer's instructions.