GSM2579537: ESC H3K9me2 ChIP-seq rep2; Mus musculus; ChIP-Seq
Murine ESCs and ESC-derived CMs were crosslinked in culture by addition of methanol-free formaldehyde (ThermoFisher; final 1% v/v) and incubated at room temperature for 10 minutes with gentle rotation. Crosslinking was quenched by addition of glycine (final 125mM) and incubated at room temperature for 5 minutes with gentle rotation. Media was discarded and replaced with PBS; cells were scraped and transferred to conical tubes and pelleted by centrifugation (250xg, 5 minutes at room temperature). Supernatant was removed, and pellets were flash frozen on dry ice and stored at -80C.    Six hours before lysing cells, 30uL protein G magnetic beads (per ChIP sample) were washed 3 times in blocking buffer (0.5% BSA in PBS); beads were resuspended in 250uL blocking buffer and 2ug antibody (Lamin B: sc6217 [Santa Cruz]; H3K9me2: ab1220 [Abcam]) and rotated at 4C for at least 6 hours. Crude nuclei were isolated from frozen crosslinked cells as follows: cell pellet (from 10cm plate) was resuspended in 10mL cold Lysis Buffer 1 (50mM HEPES-KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP‐40, 0.25% Triton X‐100, and protease inhibitors), and rotated at 4C for 10 minutes, followed by centrifugation (250xg, 5 minutes at room temperature). Supernatant was discarded and the pellet was resuspended in 10mL cold Lysis Buffer 2 (10mM Tris‐HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, and protease inhibitors), and rotated at room temperature for 10 minutes, followed by centrifugation (250xg, 5 minutes at room temperature). Supernatant was discarded and nuclei were resuspended/lysed in 1mL cold Lysis Buffer 3 (10mM Tris‐HCl, pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na‐Deoxycholate, and protease inhibitors) and transferred to pre-chilled 1mL Covaris AFA tubes (Covaris) Samples were sonicated using a Covaris S220 sonicator (high cell chromatin shearing for 15 minutes; Covaris). Lysates were transferred to tubes and Triton X-100 was added (final 1%) followed by centrifugation (top speed, 10 minutes at 4C in microcentrifuge). Supernatant was transferred to a new tube; protein concentration was measured by Bradford assay. Antibody-conjugated beads were washed 3 times in blocking buffer, resuspended in 50uL blocking buffer and added to 500ug input protein for overnight incubation with rotation at 4C. 50ug lysate was aliquoted and stored at -20C for input. On day 2, beads were washed 5 times in 1mL RIPA buffer (50mM HEPES‐KOH pH 7.5, 500mM LiCl, 1mM EDTA, 1% NP‐40, 0.7% Na‐Deoxycholate) with 2-minute incubation at room temperature with rotation for each wash. Beads were washed in 1mL final wash buffer (1xTE, 50mM NaCl) for 2 minutes with rotation at room temperature before final resuspension in 210uL elution buffer (50mM Tris‐HCl pH 8.0, 10mM EDTA, 1% SDS). To elute, beads were incubated with agitation at 65C for 30 minutes. 200uL eluate was removed to a fresh tube, and all samples (ChIP and reserved inputs) were reverse-crosslinked overnight at 65C with agitation for a minimum of 12 hours, but not more than 18 hours. 200uL 1xTE was added to reverse crosslinked DNA to dilute SDS, and samples were RNaseA treated (final 0.2mg/mL RNase; 37C for 2 hours) and Proteinase K (final 0.2mg/mL Proteinase K; 55C for 2 hours) before phenol:chloroform extraction and resuspension in 10mM Tris-HCl pH 8.0. ChIP and input DNA was quantified by Qubit (ThermoFisher) before library preparation using the NEBNext Ultra II DNA library prep kit (NEB). Samples were indexed for multiplex sequencing. Library quality was analyzed by BioAnalyzer (Agilent Genomics) and quantified using qPCR (Kapa Biosystems). Libraries were pooled for multiplex sequencing, requantified, and sequenced on the Illumina NextSeq500 platform (vII; 75bp single end sequencing; Illumina).
GEO Accession
GSM2579537