GSM4142682
GSM4142682: MeCP2_KO_HiC_rep2; Mus musculus; Hi-C
Forebrain (cortex and hippocampus) was dissected from 8-week-old MeCP2 KO and WT mice and flash frozen. In situ Hi-C was performed as described in (Rao et al., 2014) with minor modifications. Tissue was homogenized in 1% formaldehyde in PBS and rotated for 10 minutes at room temperature. Cross-linking was quenched with 125 mM glycine for 5 minutes at room temperature. Tissue was pelleted by spinning at 500 g for 5 minutes at 4°C, washed once with PBS, then spun again at 500 g for 5 minutes at 4°C. Cells were lysed by resuspending in 500 μL Hi-C lysis buffer (10 mM Tris pH 8.0, 10 mM NaCl, 0.2% Igepal CA630) with protease inhibitors (Sigma P8340), incubated on ice for 15 minutes, then spun at 2500 g for 5 minutes at 4°C. Pelleted nuclei were washed with Hi-C lysis buffer, then spun at 2500 g for 5 minutes at 4°C. Nuclei were resuspended in 50 μL 0.5% sodium dodecyl sulfate (SDS) and incubated at 62°C for 7.5 minutes. After heating, 115 μL of water and 25 μL of 10% Triton X-100 were added to quench the SDS, and samples were incubated at 37°C for 15 minutes. Restriction digest was performed by adding 25 μL of 10X NEB Buffer 2 and 100 U of MboI (NEB R0147) and rotating 16 hours at 37°C. Samples were incubated at 62°C for 20 minutes to inactivate MboI. To fill in restriction fragment overhangs and mark DNA ends with biotin, 50 μL of fill-in master mix was added (37.5 μL of 0.4 mM biotin-14-dATP, 1.5 μL of 10mM dCTP, 15 μL of 10mM dGTP, 1.5 μL of 10mM dTTP, and 8 μL 5U/μL DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210)), and samples were rotated for 75 minutes at 37°C. 900 μL of Ligation master mix was added (669 μL of water, 120 μL of 10X NEB T4 DNA ligase buffer (NEB, B0202), 100 μL 10% Triton X-100, 6 μl 20 mg/mL Bovine Serum Albumin, and 5 μL of 400 U/μL T4 DNA Ligase (NEB, M0202)), and samples were rotated at room temperature for 4 hours. Protein was degraded by adding 50 μL 20 mg/mL proteinase K (NEB, P8102) and 120 μL of 10% SDS and incubating at 55°C for 30 minutes on a Thermomixer at 350 rpm. 130 μL of 5M NaCl was added and samples were incubated at 68°C for 16 hours to reverse cross-links. DNA was purified by adding 1.6X volumes of ethanol and 0.1X volumes of 3M sodium acetate, pH 5.2 and incubated at -80°C for 15 minutes. Samples were spun at 17,720 g for 15 minutes at 4°C, washed with 70% ethanol, and spun at 17,720 g for 5 minutes at 4°C. Pellet was resuspended in 130 uL 1X Tris buffer (10 mM Tris-HCl, pH 8) and incubated at 37°C to fully dissolve DNA. To make biotinylated DNA suitable for high-throughput sequencing, DNA was sheared with a Covaris M220 (Peak incident power: 50, Duty factor: 10%, Cycles per burst: 200, Treatment time: 70 seconds). DNA fragments in the range of 300-500 bp were selected using AMPure XP beads. 150 μL of 10 mg/mL MyOne Streptavidin T1 beads were washed with 400 μL 1X Tween Washing Buffer (TWB: 5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1M NaCl, 0.05% Tween 20). Beads were resuspended in 300 μL 2X Binding Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2M NaCl), added to samples, and rotated for 15 minutes at room temperature. Beads were separated on a magnet and washed twice with 600 μL TWB, incubating at 55°C with mixing at 350 rpm for 2 minutes on a Thermomixer, and transferring to a new tube with each wash. Beads were washed once with 100ul 1X NEB T4 DNA ligase buffer, then resuspended in 100 μl of master mix (88ul 1X NEB T4 DNA ligase buffer, 2 ul 25 mM dNTP mix, 5 ul 10U/ul NEB T4 PNK (NEB, M0201), 4 ul of 3U/ul NEB T4 DNA polymerase I (NEB, M0203), and 1 ul of 5U/ul NEB DNA polymerase I, Large (Klenow) Fragment (NEB, M0210)), and incubated at room temperature for 30 minutes. Beads were washed twice with TWB as described above, washed once with 100 ul 1X NEBuffer 2, and resuspended in 100ul dATP attachment master mix (90ul 1X NEBuffer 2, 5ul 10mM dATP, and 5ul of 5U/ul NEB Klenow exo minus (NEB, M0212)), and incubated at 37°C for 30 minutes. Beads were washed twice with TWB as described above and washed once with 100ul 1X Quick ligation reaction buffer (NEB, B6058). Beads were resuspended in 50ul of 1X NEB Quick ligation reaction buffer, and 2ul NEB DNA Quick ligase (NEB, M2200) and 3 ul of NEBNext adapter (NEB E7335) diluted 1:10 in 10mM Tris pH 7.5 were added, and incubated at room temperature for 15 minutes. Beads were washed twice with TWB as described above, and washed once with 100ul 1X Tris buffer. Beads were resuspended in 20ul 1X Tris buffer and boiled at 98°C for 10 minutes to elute DNA off beads. Hi-C libraries were amplified by adding to 17 ul DNA: 25 ul NEBNext Q5 Hot Start HiFi PCR Master Mix (M0543), 3 ul NEBNext USER Enzyme (M5505), 2.5 ul 10uM Index (i7) primer, and 2.5ul Universal (i5) primer (E7335), and amplified for 8 cycles. Libraries were purified with AMPure beads (2 purifications with 0.7X beads)
GEO
GSM4142682
GEO Accession
GSM4142682