GSM6208603: Exosc3_mESC_cKO_H3K9me3_Rep2; Mus musculus; ChIP-Seq
H3K9me3: Approximately 1x107 control and Exosc3 cKO pluripotent stem cells were dissociated from culture and fixed in 50 mL of N2B27 media with 1% methanol free formaldehyde for 10 minutes at room temperature. Fixation was quenched using .125 M glycine for 5 mins at room temperature. Pellets were then washed 3x with ice-cold DPBS supplemented with Halt protease and phosphatase inhibitors (Thermo Scientific, 78446). Pellets were frozen in -80°C overnight. Frozen pellets were then thawed on ice and 10x106 cells were lysed with 1 mL of LB1 (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton x-100) for 20 minutes rotating in 4°C. Chromatin was pelleted and supernatant was then discarded. Nuclear envelope of the chromatin fraction was lysed with 1 mL LB2 (Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 M EGTA), rotating for 10 minutes at room temperature. Pellets after LB2 lysis were resuspended in 300 µL of LB3 (Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 M EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine). Chromatin was sonicated in LB3 was aliquoted in 100 µL increments in micro AFA Fiber Crimp-Cap 6x16mm tubes (Covaris, 520091). Chromatin was sheared on a Covaris instrument with peak power set to 450 for 700 seconds. Aliquots of sheared chromatin were pooled for immunoprecipitation. Antibody was coupled to Protein A Dynabeads (Invitrogen, 10001D) for 4 hours rotating in 4°C. Coupled beads were washed 3x with 0.05% BSA in DPBS. Antibody bound beads were then added to chromatin in LB3 overnight rotating in 4°C. Beads were washed 8 times with RIPA buffer (500 mM Hepes-KOH pH 7.6, 100 mM LiCl, 0.5 M EDTA, 1% NP-40, 0.7% Na- Deoxycholate). An additional wash with TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 50 mM NaCl) prior to elution. Bound chromatin was eluted in elution buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS) shaking for 30 minutes at 65°C. Decrosslinking was achieved by the addition of 8 µL 5 M NaCl to 210 µL of eluted chromatin. RNA and protein were digested by using 500 µg/mL RNase (Invitrogen, AM2271) and 22 mg/mL proteinase K (Invitrogen, AM2546) for 2 hours at 55°C. DNA for library preparation and sequencing was purified using MinElute PCR Purification Kit (Qiagen, 28004).     H3K27ac and Rbp1: Cell fixation and quenching were done exactly as described above. Approximately 10x106 cells were lysed for 15 minutes on ice in 500 µL of cell lysis buffer 1 (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% NP-40). Nuclei were pelleted and subsequently lysed in 500 µL of nuclear lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS) for 10 minutes on ice. Chromatin was then sheared using the Diagnode Bioruptor Plus for 30 cycles set to 30 seconds on and 30 seconds off. Sheared chromatin was then precleared using species specific IgG for 2 hours at 4°C. Antibodies for protein of interests bound to chromatin were couple to Protein A Dynabeads (Invitrogen, 10001D). Coupled beads were added to precleared chromatin and rotated overnight at 4°C. Beads bound to chromatin were then resuspended with 1 mL of IP wash I buffer (20 mM Tris-HCl pH 8, 2 mM EDTA, 50 mM NaCl, 1% Triton x-100, 0.01%SDS). Beads were bound to a magnetic stand and washed twice with a high salt buffer (20 mM Tris-pH 8, 2 mM EDTA, 500 mM NaCL, 1% Triton x-100, 0.01% SDS0 and once with IP wash II buffer (10 mM Tris ph8, 1 mM EDTA, 0.25 M LiCl, 1% NP-40, 1 % Na- Deoxycholate). DNA was eluted twice by applying 100 µL of elution buffer (1.25% SDS, 100 mM NaHCO3) to each sample and incubating at 65°C shaking at 800 rpm. Decrosslinking and DNA purification were identical to the steps described above. Paired-end libraries from purified DNA were prepared using NEBNext® Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S) per manufacturer recommendations. Size selection of DNA prior to library was not performed.