Computational protocol: Megakaryocyte lineage development is controlled by modulation of protein acetylation

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Protocol publication

[…] CD34+ cells were differentiated towards megakaryocytes for 4 days, followed by overnight treatment with VPA, or NAM. Lysates were prepared, and ChIP was performed as described previously [] utilising an anti-acetylated H3K27 antibody (ab4729, Abcam, Cambridge, MA, USA). Next, chromatin was sheared, end-repaired, followed by ligation of sequencing adaptors, amplification of the library by ligation–mediated PCR (LMPCR). After LMPCR, the library was purified, checked for the proper size range, and the absence of adaptor dimers on a 2% agarose gel, followed by sequencing on the SOLiD/AB sequencer (Applied Biosystems Life Technologies, Carlsbad, CA, USA). Sequencing reads were mapped against the reference genome (hg19,NCBI3) using the BWA package (-c–l 25 –k 2 –n 10) []. Non-uniquely placed reads were discarded. Cisgenome v2.0 software package [] was used for the peak calling from the ChIP-seq data with settings–e 50 –maxgap and further analysis. Cisgenome 2 was used with settings: -e 50, -maxgap 200 and -minlen 200. A combination of Cisgenome functions, custom PERL and R scripts was used for additional data analysis. Data were normalised for the total amount of acetylation. […]

Pipeline specifications

Software tools BWA, CisGenome
Application ChIP-seq analysis
Organisms Homo sapiens
Chemicals Niacinamide, Valproic Acid