title: GSM1873366: CARM1; Homo sapiens; ChIP-Seq
library_construction_protocol: The starting materials are two 15 cm plates of MCF-7 cells for each ChIP. When cells reached a >90% confluent monolayer, 1% formaldehyde was added in culture media to fix cells for 10 minutes at 37°C. Cells were washed twice using ice cold PBS and then pelleted for nuclei purification using Nuclei Isolation Kit (Sigma, Nuc 101). Nuclei were lysed with SDS lysis buffer and chromatin was sonicated using a Bioruptor (Diagenode, B01020001) at high power with 6 pulses of 30 seconds sonication and 30 seconds rest to obtain DNA fragments of optimum size (between 200 bp and 1 kb). The remaining steps were performed using the protocol from Chromatin Immunoprecipitation (ChIP) Assay Kit (Millipore, 17-295). The eluted DNA was further purified using the QIAquick PCR Gel Extraction Kit (Qiagen,28706 ) for ChIP-seq analysis. The Illumina compatible indexed libraries were prepared using KAPA Library preparation kit, as per the manufacturer’s instructions. In brief, DNA was fragmented to a median size of 150bp by sonication. Fragmented DNA ends were polished and 5′-phosphorylated. After addition of 3′-A to the ends, indexed Y-adapters were ligated and the samples were PCR amplified. The resulting DNA libraries were quantified and validated by qPCR, then sequenced 6 samples per lane on Illumina’s HiSeq2000 sequencer using the 36 nt single-read format. The resulting BCL files containing the sequence data were converted into “.fastq.gz” files and individual sample libraries were demultiplexed using CASAVA 1.8.2 with no mismatches.
attributes.tag: GEO Accession
attributes.value: GSM1873366