GSM8067842
Cells were crosslinked in 1% v/v formaldehyde for ten minutes and quenched by adding 2.5 M glycine to a final concentration of 0.2 M for 5 min. Cells were pelleted by centrifugation at 300 G for 5 min at 4 °C and then washed with cold PBS. Cells were lysed with lysis buffer containing 10 mM Tri-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA630 and protease inhibitors for 15 min on ice. Cells were centrifuged at 300 G and washed in that same buffer, and then resuspended in 50 µl 0.5% SDS for 5 min at 62 °C. Afterward, 145 µl of water and 25 µL of Trition X-100 were added and incubated at 37 °C for 15 min. Chromatin was digested overnight in MboI, MseI, or NlaIII in the corresponding buffer. Fragment overhangs were repaired and biotinylated using equal amounts of biotin-14-dATP, dCTP, dGTP, and dTTP in the presence of 40 units of DNA Polymerase I, Large (Klenow) Fragment at 37 °C for 1 h. Chromatin was ligated in 1x T4 DNA ligase buffer, Triton X-100, 0.12 mg BSA, and 2000 units T4 DNA ligase at room temperature for four hours. Proteins were digested, and chromatin decrosslinked in 0.72 mg/ml proteinase k, 1% SDS, and 0.5 M NaCl for 30 min at 55 °C, followed by 68 °C overnight. Libraries were sequenced and then processed using Illumina HiSeq 4000 software and JuicerTools v1.14.08. As per Harris, Hannah L., et al. "Chromatin alternates between A and B compartments at kilobase scale for subgenic organization." Nature Communications 14.1 (2023): 3303.