GSM4388143: MOLM13_HiC_rep1; Homo sapiens; Hi-C
In brief, 2 x 107 MOLM-13 human AML cells were fixed with 1% formaldehyde for 10 minutes at room temperature, followed by immediate addition of 2.5M glycine. The cell pellet in 1 ml of ice-cold lysis buffer (10 mM Tris-HCl, pH = 8.0, 10 mL NaCL, 0.8% NP40) containing protease inhibitor cocktail (Thermo Scientific) and incubated at 15 minutes on ice prior to homogenization with a tight Dounce pestle. To open chromatin, the homogenized lysate was then subjected to 0.1% SDS and incubated for 10 minutes at 65 °C to open chromatin, followed by addition of Triton-X (1 % final concentration. Chromatin was then digested overnight at 65 °C with HindII (Thermo Scientific). Digested DNA ends were then filled in with biotin-14-dCTP, as well as non-biotinylated dATP, dGTP and dTTP using Klenow (Invitrogen). Blunt end proximity ligation was performed using T4 DNA ligase (Invitrogen) for 4 hours at 16 °C, followed by overnight proteinase K treatment to remove DNA-bound proteins. Proximity-ligated DNA was purified using phenol chloroform DNA purification and subjected to the removal of biotin from un-ligated DNA ends. DNA was purified using Amicon Ultra 30K spin columns (Millipore) and sonicated to produce 150-350 DNA fragments using the Covaris instrument (Duty Cycle 10%, Intensity 5, Cycles per Burst 200, Frequency Sweeping mode with contant degassing for 3 cycles and 60 seconds process time). Sonicated DNA was purified using AMP pure XP beads (0.8X large fragment exclusion step followed by 1.1X bead bound purification). End repair of purified sonicated DNA was performed using T4 polynucleotide kinase and Klenow DNA polymerase, followed by dATP-tailing using Klenow fragment (3'-5' exo). Biotinylated Hi-C DNA was then enriched using MyOne Streptavidin C1 beads, followed by Illumina paired-end adapter ligation and PCR amplification (Q5 HotStart) under the following conditions: : (Step 1) 98°C for 30 seconds, (Step 2) 98°C for 30 seconds, (Step 3) 63°C for 30 seconds, (Step 4) 72°C for 1 minute, and (Step 5) 4°C hold, with amplified library purified using AMP pure bead cleanup (1.5x bead reaction). For cycling PCR steps 2-4, the number of cycles used ensured PCR amplification remained in the linear range with the expected library size distribution as assessed by agarose gel visualization.
GEO Accession
GSM4388143