GSM9009690: MCD, Splenic CD8+ T cells, Hi-C, rep 1; Mus musculus; Hi-C
10 million splenic CD8+ T cells in each sample were isolated and resuspended in 10 ml complete medium (80% RPMI 1640 and 20% FBS) and fixed with 571 μl 37% Formaldehyde (F8775, Sigma) for 10 min at room temperature, and 894 μl 2.5 M Glycine (7005, CST) was added and mixed to neutralize formaldehyde for 10 min at room temperature and another 15 min on ice. 10 million splenic CD8+ T cells in each sample were isolated and resuspended in 10 ml complete medium (80% RPMI 1640 and 20% FBS) and fixed with 571 μl 37% Formaldehyde (F8775, Sigma) for 10 min at room temperature, and 894 μl 2.5 M Glycine (7005, CST) was added and mixed to neutralize formaldehyde for 10 min at room temperature and another 15 min on ice. The samples were centrifuged at 400 g for 5 min at 4 ℃ and the cell pellets were washed, pelleted and flash frozen on dry ice for storage in -80 ℃ before Hi-C protocol was initiated. Briefly, cells were lysed using lysis buffer with protease inhibitors. After the completion crosslinking and lysis, chromatin was digested with 200 U DPN Ⅱ restriction enzyme at 37 ℃. The DNA ends were labeled with biotin and incubated for 45 min at 37 ℃, and then, the enzyme was inactivated at high temperature. DNA ligation was performed by T4 DNA ligase and incubated for 2 h. After this, the proteinase K was added to reverse cross-linking for 2 h at 65 ℃. DNA fragments were purified and dissolved in water and fragmented into 350-500 bp, and the DNA ends were repaired. DNA fragments labeled by biotin were finally separated by Dynabeads M-280 Streptavidin. Hi-C libraries were controlled for quality and sequenced on Illumina Novseq platform at Novogene Science and Technology (Beijing, China).