Illumina HiSeq 1500 sequencing; ChIP-seq of FLAG-tagged SMARCAD1 in female mouse embryonic stem cells (PGK12.1 XX)
ChIP-seq of FLAG-tagged SMARCAD1 in female mouse embryonic stem cells (PGK12.1 XX)
Approximately 5x10e7 PGK12.1XX 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). Mouse PGK12.1 XX ES cells were cultivated feeder-cell free on gelatin-coated dishes in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies) supplemented with 15% fetal calf serum, 2 mM L-glutamine, 1x non-essential amino acids, 50 μM 2-mercaptoethanol, and 1000U/ml ESGRO leukemia inhibitory factor. Generation of FLAG-SMARCAD1 expressing PGK12.1 XX ES cells: The coding sequence of SMARCAD1 was amplified from mouse ES cell cDNA and inserted into a chicken beta actin promoter (CAG)-driven expression vector, with an N-terminal triple FLAG tag. Tagged wild-type Smarcad1 construct along with the empty FLAG vector control were transfected into PGK12.1 ES cells using Lipofectamine 2000 (Life Technologies), followed by 1.7 µg/ml puromycin selection and clonal expansion. Chromatin was eluted in 1% SDS, 100mM NaHCO3 and DNA purified by Qiaquick PCR cleanup columns (Qiagen). Library preparation was performed using the MicroPlex Library Preparation Kit v2 (Diagenode) according to manufacturer's instructions. Briefly: 7ng DNA was used for each sample. Adapter-ligated DNA was subject to 9 cycles of PCR amplification before size selection and DNA purification by AMPure XP beads (Agencourt).
Philipps-University Marburg, Genomics Core Facility
E-MTAB-7014:flag_ChIP_flagSMARCAD1_s
Experimental Factor: immunoprecipitate
anti-flag (F1804, Sigma-Aldrich)
Experimental Factor: genotype
SMARCAD1-3xFLAG