H3K27ac (Cell Signaling Technology, Cat #8173, lot 1)
developmental stage
Embryo
strain
C57BL/6
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed with 1% formaldehyde for 10 mins, and followed by glycine for 5 mins. Chromatin DNA was sheared to 200–500 bp average in size by sonication and chromatin was immunoprecipitated with antibodies. Anti Oct4, Sox2, Nanog, Wdr5, H3K27ac, and Med1 were used to capture protein-DNA complex. Protein A/G magnetic beads (Dynabeads) were added and incubated overnight at 4°C. After washing and elution, the protein–DNA complex was reverse-crosslinked by heating at 65°C, and immunoprecipitated DNA was purified by using QIAquick Spin column. ChIP-seq libraries were prepared using the NEBNext® Ultra™II DNA Library Prep Kit. Libraries were sequenced on the Illumina X system following the manufacturer's protocols. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). 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 Genome Analyzer following the manufacturer's protocols.