Illumina HiSeq 1500 sequencing; ChIP-Seq of chromatin remodeler SMARCAD1 and H3K9me3 in female mouse embryonic stem cells (PGK12.1 XX)
ChIP-Seq of chromatin remodeler SMARCAD1 and H3K9me3 in female mouse embryonic stem cells (PGK12.1 XX)
Approximately 5x10e7 PGK12.1XX (CVCL_AS07) cells per 15 cm plate were cross-linked with 2 mM DSG (disuccinimidyl glutarate, Thermo Scientific) in PBS for 45 min at room temperature, washed three times with PBS, and formaldehyde (Polysciences) was added to a final concentration of 1 % for 10 min at room temperature. Formaldehyde was quenched using glycine (0.125 M). Antibodies used were SMARCAD1 PAB15737 (Abnova) and H3K9me3 ab8898 (abcam). Mouse PGK12.1 ES cells were cultivated feeder-cell free on gelatin-coated dishes in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies) supplemented with 15% foetal calf serum, 2 mM L-glutamine, 1x non-essential amino acids, 50 μM 2-mercaptoethanol, and 1000U/ml ESGRO leukemia inhibitory factor. Generation of SMARCAD1 and control knockdown ES cells was achieved by transfecting shRNA vectors targeting exon 7 of Smarcad1 (shSMARCAD1) or a linker sequence (shControl) as control into PGK12.1 XX cells using Lipofectamine 2000 (Life Technologies), followed by puromycin selection and clonal expansion. Chromatin was eluted in 1% SDS, 100mM NaHCO3 and DNA purified by Qiaquick PCR cleanup columns (Qiagen). Five individual SMARCAD1 ChIPs were pooled and purifed on QIAquick columns (Qiagen). Four nanograms of SMARCAD1 and H3K9me3 antibody precipitated DNA were used for library preparation with the Microplex library preparation kit v2 (Diagenode). Libraries were purified on AMPure magnetic beads (Beckman Coulter).
Philipps-University Marburg, Genomics Core Facility
E-MTAB-7012:endogenous_SMARCAD1_ChIP_wt_s
Experimental Factor: immunoprecipitate
anti-SMARCAD1
Experimental Factor: RNA interference
scrambled control