4 protocols
Base-calling: CASAVA_1.6 Alignment: Bowtie. Genome build mm9. Peak-calling: Genomic stretches at a resolution of 50bp showing coverage in the top 0.1% of the genome for P300 or RUNX1 and not showing NIS in the top 1% of the genome. Genome_build: MGSCv37
Murine FL-derived mature MKs were produced essentially as previously described by Shivdasani RA, Schulze H (2005) Culture, expansion, and differentiation of murine megakaryocytes. Curr Protoc Immunol Chapter 22: Unit 22F 26. Briefly, FL cells were derived from E14.5 embryos and cultured in DMEM medium supplemented with 10% FBS (Gibco, US), 2mM L-glutamine and penicillin/streptomycin and TPO (50ng ml-1) at 37ºC and 10% CO2. After 7 days in culture, mature MKs were isolated by two sequential 2%-4% BSA gradients.
Culture in DMEM.
nucleic acid library construction protocol
ChIP samples were prepared using 5*10^7 cells per sample. Chromatin was prepared as described in Ainbinder et. Al Mol Cell Biol. 2002 (PMID 12192035). Antibodies used: NIS - non-immune serum was taken from our rabbits; RUNX1- our home-made antibody, described in Aziz-Aloya RB, Levanon D, Karn H, et al. Expression of AML1-d, a short human AML1 isoform, in embryonic stem cells suppresses in vivo tumor growth and differentiation. Cell Death Differ. 1998;5(9):765-773 (PMID 10200536); and P300 - C-20:rabbit polyclonal IgG, Santa Cruz Biotechnology sc-585, lot #E2010. ChIP libraries were created following the Illumina sample preparation protocol from 10ng of DNA. Following end-repair and A-base addition, primers were ligated and samples were size selected on a 2% agarose gel. Fragments of 200bp were excised and purified using the Qiagen gel-extraction kit with gel melting at room temperature. Enrichment was carried out using 18 cycles of amplification. Library yield was quantified using Qubit (Invitrogen), while final fragment sizes were determined using Agilent BioAnalyzer. The ChIP-seq libraries were run on an Illumina GAIIx at the Department of Biological Services at WIS, using Illumina cluster generation kits and sequencing reagents.