title: GSM3500785: D3-8_input_Rep2; Mus musculus; ChIP-Seq
library_construction_protocol: Chromatin immunoprecipitation was done according to a published method (O'Geen, H., Nicolet, C.M., Blahnik, K., Green, R. & Farnham, P.J. Comparison of sample preparation methods for ChIP-chip assays. Biotechniques 41, 557-580 (2006). Briefly, mESCs were crosslinked with 1% formaldehyde in PBS for 10 min at room temperature and the reaction was stopped by adding glycine to final concentration of 1.25M for 5 minutes at room temperature. Crosslinked cells were collected with a cell scraper, rinsed twice with PBS, and resuspended at a concentration of 5 x 10^7 cells/mL of swelling buffer (0.1 M Tris pH 7.6, 10 mM KOAc, 15 mM MgOAc, 1% NP40) to remove the cytoplasmic fraction. Nuclei were resuspended at a concentration of 10 x 10^8 cells/mL of nuclei lysis buffer (50 mM Tris-Cl pH 8.0, 10 mM EDTA, 1% SDS), and lysate was sonicated for 90 cycles (30 sec on / 50 sec off) in a Diagenode Bioruptor (Diagenode UCD-300). The sonicated chromatin was precleared by adding washed and blocked StaphA at a concentration of 10 uL/1 x 10^7 cells (Calbiochem 507862). StaphA were prepared as previously described, washed in dialysis buffer (2 mM EDTA, 50 mM Tris-Cl pH 8.0), and blocked in 10 mg/mL salmon sperm DNA (EMD Millipore 16-157) and 10 mg/mL BSA. For each IP, 100 uL of precleared chromatin was diluted in twice the volume of IP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.0, 167 mM NaCl) and 5 ug of antibody was added. Antibodies against H3K4me3 (Abcam ab8580), H3K9Ac (Active Motif 39137) were used. Libraries were constructed using the NEBNext DNA Sample Prep Master Mix Set for Illumina (NEBNext E6040) and NEBNext Multiplex Oligos for Illumina (NEBNext E7335 and E7500), according to kit instructions. Libraries were then quantified using the KAPA Library Quantification Kit with Universal qPCR Master Mix (KAPA Biosystems, KK4824) according to kit instructions, and quantitative PCR was performed using the SYBR GreenER MasterMix (Invitrogen) on an ABI 7900HT Detection System. Pooled samples were sequenced on a HiSeq4000.
attributes.tag: GEO Accession
attributes.value: GSM3500785