4 protocols
normalization data transformation protocol
Basecalls performed using CASAVA version 1.4 ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie Only reads that aligned to a unique position in the genome with no more than two sequence mismatches were used for further analysis. The number of unique reads was calculated in bins across the genome. Bins containing statistically significant Chip-seq enrichment were identified by comparison to a Poissonian background model. Genome_build: mm9 Supplementary_files_format_and_content: wig file contains the p-values of the bins across the genome
sample treatment protocol
ES cells were allowed to aggregate and differentiate to form embryoid bodies by removing Leukemia inhibitory factor (LIF) from culture medium
growth protocol
mouse ES cells were routinely passaged on irradiated mouse embryonic fibroblasts using medium containing Knock-out Dulbecco’s modified Eagle’s medium (KO-DMEM; GIBCO), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone), 0.055 mM β-mercaptoethanol (GIBCO), 2 mM L-glutamine (GIBCO), 0.1 mM nonessential amino acid (GIBCO), 5000 U/mL penicillin-streptomycin (GIBCO), 15mM HEPES (GIBCO) and 1000 U/mL LIF (Millipore/Chemicon).
nucleic acid library construction protocol
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. 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.