Illumina HiSeq 1500 sequencing; ChIP-Seq of SMARCAD1 and KAP1 in male mouse embryonic stem cells (E14)
ChIP-Seq of SMARCAD1 and KAP1 in male mouse embryonic stem cells (E14)
KAP1 antibody, ab22553  (abcam), was used in inducible SMARCAD1 knockdown cells prior to and after two day induction of SMARCAD1 knockdown as well as in SMARCAD1 knockdown cells that express a FLAG tagged SMARCAD1 transgene; either wild-type or an ATPase mutant. FLAG antibody used was F1804 (Sigma) in SMARCAD1 knockdown cells expressing no FLAG tagged protein (negative control) and in cells expressing FLAG tagged SMARCAD1; either wild-type or an ATPase mutant. Mouse E14 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, penicillin/streptomycin and 1000U/ml ESGRO leukemia inhibitory factor. To generate an enzymatically inactive SMARCAD1 chromatin remodeler a mutation in the ATP binding pocket (K523R) was generated by site-directed mutagenesis using the QuikChange Lighting Site Directed Mutagenesis Kit (Agilent 210518) and the following oligos: Fwd 5'-GCAGACGAAATGGGCCTAGGAAGAACCATTCAAGCCATTGC -3' Rev 5'- GCAATGGCTTGAATGGTTCTTCCTAGGCCCATTTCGTCTGC-3'. A Triple-FLAG tagged SMARCAD1 wild-type (E14 S-KD.WT) and ATPase mutant (E14 S-KD.ATPase_mt) constructs were stably integrated into E14 cells carrying a doxycycline inducible shRNA (E14.) targeting the 3'UTR of SMARCAD1 (shSMARCAD1) as described (Ding et al., 2017) prior to knocking down the endogenous SMARCAD1 (E14 S-KD).  Doxycycline (0.5µg/ml) treatment was performed for 2 days to achieve knockdown of endogenous SMARCAD1 before ChIP was carried out. E14 chromatin cross-linking was performed 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). Chromatin was eluted in 1% SDS, 100mM NaHCO3 and DNA purified by Qiaquick PCR cleanup columns (Qiagen). Six (FLAG) or four (KAP1) individual ChIPs were pooled and purifed on QIAquick columns (Qiagen). Five nanograms of precipitated DNA were used for library preparation using the Microplex library preparation kit v2 (Diagenode). Libraries were purified on AMPure magnetic beads (Beckman Coulter).
Philipps-University Marburg, Genomics Core Facility
E-MTAB-7011:E14.S-KD.wt_FLAG_s
Experimental Factor: immunoprecipitate
anti-flag (F1804, Sigma-Aldrich)
Experimental Factor: RNA interference
endogenous SMARCAD1 knockdown by shRNA
Experimental Factor: phenotype
SMARCAD1-3xFLAG
Experimental Factor: compound
doxycycline
Experimental Factor: dose
0.5