title: GSM2897814: Input; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were fixed for 15 min with 1/10 volume of freshly prepared formaldehyde solution (11% formaldehyde, 0.1 M NaCl, 1 mM EDTA, and 50 mM HEPES). The fixation was stopped by adding 1/20 volume 2.5 M glycine for 5 min. Fixed HESCs were collected and pelleted at 800 x g for 10 min at 4 oC. Cell pellets were washed two times with cold PBS-Igepal (0.5% Igepal, 1 mM PMSF).  HESCs from six patients were pooled before genomic DNA isolation. NR2F2 immunoprecipitation and DNA library generation were performed by Active Motif. ChIP and input DNA were amplified using the Illumina ChIP-Seq DNA Sample Prep Kit. Briefly, DNA ends were polished and 59-phosphorylated using T4 DNA polymerase, Klenow polymerase, and T4 polynucleotide kinase. Addition of 3'-adenine to blunt ends using Klenow fragment (3'-5' exo minus), Illumina genomic adapters were ligated and the sample was size fractionated to 175–225 bp on a 2% agarose gel. After amplification for 18 cycles with Phusion polymerase, the resulting DNA libraries were tested by RT-qPCR at the same specific genomic regions as the original ChIP DNA to assess quality of the amplification reactions.
attributes.tag: GEO Accession
attributes.value: GSM2897814