Day2 embryoid bodies induced with dox for another 36h
chip-antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For Chromatin Immunoprecipitation-sequencing (CHIP-seq), day2 EBs were induced with Dox for 36h and fixed in 1% formaldehyde solution for 15 min and quenched with 0.125M Glycine and flash-frozen. Sonicated nuclear extracts were incubated with anti-Flag M2 antibody (Sigma) and antibody-chromatin complexes were extracted using prepared Staph A cells (Pansorbin, Calbiochem). Libraries were prepared from immunoprecipitated DNA using illumina Chip-seq DNA sample prep kit (illumina) according to manufacturer’s instructions. Immunoprecipitated enriched DNA was end repaired converting the overhangs into phosphorylated blunt ends, using T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase (PNK). To the blunt phosphorylated 3’ end of DNA Fragments, ‘A’ base was added using Klenow fragment (3’ to 5’ exo minus) allowing the ligation of DNA fragments to adapters, which have a single ‘T’ base overhang at their 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. DNA libraries were sequenced on Genome Analyzer II according to manufacturer’s instructions.