title: GSM4662098: H3k9me2 chip in MEF cells 48h in reprogramming (+DOX + C3 + UNC0638) - replicate2; Mus musculus; ChIP-Seq
library_construction_protocol: Native ChiIP-seq was performed as previously described (Chen et al., 2018). 10 million cells were trypsinized, washed in ice-cold PBS, and flash frozen. Cell pellets were resuspended in Dounce Buffer (10mM Tris-HCl pH 7.5, 4mM MgCl2, 1mM CaCl2, protease inhibitors) with a 1:1 ratio of Drosophila Kc cells (for spike-in controls) and homogenized using 20 repetitions of a 25G syringe. Cells were treated with 150U/ml micrococcal nuclease (Worthington Biochemical) for 20 minutes at 37C and reactions were quenched with 0.5M EDTA and incubated on ice for 5 minutes. MNase-digested cells were incubated on ice for 1 hour with vortexing every 10 minutes with 1ml Hypotonic Lysis Buffer (0.2mM EDTA, 0.1mM benzamidine, 0.1mM PMSF, 1.5mM DTT, PIC) followed by a 3000g spin at 4C for 5 minutes. Supernatants were precleared by rotation for 2 hours at 4C with Protein G Dynabeads (Life Technologies) that had been washed 3x in IP Buffer (10mM Tris-HCl pH 8.0, 1% Triton X-100, 0.1% Deoxycholate, 0.1% SDS, 90mM NaCl, 2mM EDTA, PIC). Precleared chromatin was combined in 2 million cell equivalents with 5ul H3K9me2 antibody (Abcam, ab1220) and 20ul washed Protein G Dynabeads in IP Buffer and rotated at 4C overnight. Beads were then washed twice with ChIP Wash Buffer (20mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2mM EDTA, 150mM NaCl, PIC), once with Final ChIP Wash Buffer (20mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2mM EDTA, 500mM NaCl, PIC), and eluted twice 100ul Elution Buffer (100mM NaHCO3, 1% SDS) and RNase A at 68C. Input chromatin for each sample was diluted to 200ul in Elution Buffer and DNA from all samples and inputs was purified using the QIAquick PCR Purification Kit (Qiagen). Libraries were prepared using the KAPA HyperPrep Kit (Roche)
attributes.tag: GEO Accession
attributes.value: GSM4662098