GSM8172225: 3T3_CDX2, HiC, Dox24h, rep1; Mus musculus; Hi-C
Two million cells were fixed in 2% formaldehyde solution (AppliChem, catalog no. UN2209), and the reaction was quenched with 2 µM glycine (VWR Chemicals, catalog no. 101196X). Cells were incubated in lysis buffer (10 mM Tris-HCl, pH 8.0 (Invitrogen, Thermo Fisher Scientific, catalog no. 15568025), 10 mM NaCl (Sigma-Aldrich, catalog no. 59222C), 0.2% IGEPAL CA-630 (Sigma-Aldrich, catalog no. I8896), 1× cOmplete Protease Inhibitor Cocktail (Sigma-Aldrich, catalog no. 11697498001)) for 30 min at 4 °C. The nuclei were spun down, resuspended in NEB3.1 buffer (New England Biolabs, catalog no. B7203S) and 0.1% sodium dodecylsulfate (SDS; Carl Roth, catalog no. CN30) final concentration, and incubated at 65 °C for 10 min. The SDS was quenched by the addition of 1% Triton X-100 (AppliChem, catalog no. A1388). The chromatin was then digested with 100 U of restriction endonuclease MboI (New England Biolabs, catalog no. R0147) overnight at 37 °C. Digested fragments were filled in with 0.4 mM biotin (Invitrogen, Thermo Fisher Scientific, catalog no. 19524016), 10 mM dCTP, 10 mM dGTP, 10 mM TTP (Promega, catalog no. U1330) using 50 U µl−1 of DNA polymerase I, large Klenow fragment (New England Biolabs, catalog no. M0210) and incubated at room temperature for 4 h. Proximity ligation of the biotin-filled ends was performed by the addition of 5 U µl−1 of T4 DNA Ligase (Thermo Fisher Scientific, catalog no. EL0011), 0.5× bovine serum albumin (New England Biolabs, catalog no. B9000S) and 1% Triton X-100 to the samples, which were incubated at room temperature for an additional 4 h. Crosslinks were reversed by treatment with 300 mM NaCl and 1% SDS overnight at 68 °C. Samples were treated with 50 µg ml−1 of RNase A (Thermo Fisher Scientific, catalog no. EN0531) for 30 min at 37 °C, followed by incubation with 400 µg ml−1 of proteinase K (Promega, catalog no. V3021) at 65 °C for 1 h. DNA was precipitated with 1.6× volumes of 100% ethanol and 0.1× volume of 3 M sodium acetate, pH 5.2 (Thermo Fisher Scientific, catalog no. R1181) and incubation at −80 °C for 1 h. DNA was purified with 70% ethanol and fragmented by sonication (Covaris E220). DNA was double size selected with 0.575×, followed by 1.2×, volumes of Ampure XP beads (Beckman Coulter, catalog no. A6388) to obtain fragments sizes of 300–700 bp. Biotin-bound fragments were isolated on Dynabeads MyOne Streptavidin T1 beads (Thermo Fisher Scientific, catalog no. 65602) by incubation in binding buffer (10 mM Tris-HCl, pH 7.5 (Thermo Fisher Scientific, catalog no. 15567027), 1 mM ethylenediaminetetraacetic acid (Invitrogen, Thermo Fisher Scientific, catalog no. AM9260G), 2 M NaCl) for 30 min at room temperature. Bead-bound DNA is subjected to end polishing though the addition of 25 mM each of a dNTP mix, 3 U µl−1 of T4 DNA polymerase, 5 U µl−1 of DNA polymerase I, large Klenow fragment, 10 U µl−1 of T4 polynucleotide kinase in 1× T4 DNA ligase reaction buffer, and incubation for 30 min at room temperature. The dA tail was added to the fragments by incubation with 10 mM dATP and 5 U µl−1 of Klenow fragment 3′→5′ exonuclease for 30 min at 37 °C. Illumina Indexed TruSeq adapters were ligated to the DNA fragments with T4 DNA ligase and incubated at room temperature for 2 h. Finally, the bead-bound libraries were amplified using KAPA HiFi HotStart ReadyMix PCR Kit, and universal Illumina forward and reverse primers.