title: GSM9042058: ESCs, rep2; Mus musculus; Hi-C
library_construction_protocol: mouse embryonic stem cells, including wild type, Sall4-KO, and SiTotiSCs of Sall4-KO, were crosslinked with 2% formaldehyde (Sigma, 47608) for 10 min at room temperature, followed with 2 mM ethylene glycol-bis (Thermo, 21565) for 45 min at room temperature twice. Each sample had two replicate libraries. For each individual library, 50,000 crosslinked cells were lysed for 1 h on ice in lysis buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and 1× protease inhibitor cocktail). Then, the supernatant was removed and cell pellets were resuspended in 0.01% SDS to incubate for 25 min at room temperature, then quenched with 3% Triton X-100. The chromatin was digested with AluI (NEB, R0137L) and HpyCH4V (NEB, R0620L) at 37 °C overnight. A-tailing of the digestion ends was achieved by dATP (NEB, N0440S), Klenow Large fragment (3'→5' exo-) (NEB, M0212L), and BSA (NEB, B9000S) for 1 h at 37 °C. The reaction was deactivated by incubating for 20 min at 65 °C. Proximity fragments were ligated at 16 °C overnight by adding Quick ligation buffer (NEB, B6058S), T4 DNA ligase (NEB, M0202L), bridge linker (Forward strand: 5'-/5Phos/TAGGCGCTGG/iBIOdT/TGCGTTGT-3', reverse strand: 5'-/5Phos/CAACGCAACCAGCGCCTAT-3'), and ddH2O. The supernatant was discarded, then pellets were resuspended in proteinase K (Thermo, AM2548) and SDS for reverse-crosslinking by shaking for 2 h at 65 °C. The DNAs were extracted using DNA Clean & Concentrator-5 (Zymo, 4013), then sonicated so that all fragments were below 500 bp. DNA products were purified using 1.6× SPRIselect beads (Beckman Coulter, B23318) and eluted with 50 μL Buffer EB (Qiagen, 19086). 50 μL previously blocked Dynabeads M-280 Streptavidin (Invitrogen, 11206D) were added to the elution, and the mixture was incubated for 45 min at room temperature. Beads were washed and resuspended with 50 μL Buffer EB. Libraries were constructed using NEBNext Ultra II DNA Library Prep Kit (NEB, E7645L). Briefly, the fragments enriched on M-280 beads went through end repair, dA-tailing, adaptor ligation, then amplification with 8 cycles using VAHTS Multiplex Oligos set 4 for Illumina (Vazyme, N321). The supernatant of PCR products was transferred with the help of magnetic rack, and purified with 1× SPRIselect beads. The final libraries were sequenced on a BGI T7 platform with 150 bp paired-end reads (PE150).