title: GSM4989588: MDA-MB-231-P3 cells-NKRF-siUc003xsl.1-treated_rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: A total of 5×10e6 to 1 x 10e7 cells transfected with siRNAs or plasmids were collected and crosslinked in 1% formaldehyde solution for 10 min at room temperature, followed by the addition of 125 mM glycine for 5 min. ChIP assay was performed with a EZ-Magna ChIP Kit (Merck Millipore, USA) following the manufacturer′s instructions. The nuclear lysis was sheared to yield DNA fragments ranging from 100 to 500 bp by a sonication system-Bioruptor (Diagenode, Belgium). Two micrograms of normal IgG were used as the negative control, and anti-NKRF (ab168829, Abcam, USA) antibodies were used for each immunoprecipitation. The IPs were eluted and reverse crosslinked, after which the DNA fragments were purified. Immunoprecipitated and input DNAs were subjected to ChIP-seq analysis. Libraries were prepared according to Illumina's instructions accompanying the Paired-EndDNA Sample PrepKit (PE-102-1001). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the BGISEQ-500 following the manufacturer's protocols.
attributes.tag: GEO Accession
attributes.value: GSM4989588