Ab4729

ChIP-seq data sets of epigenetic marks associated with active enhancers were analyzed to identify enhancer sequences in H9-ESC that were differentiated into inhibitory interneurons (Eshel et al., unpublished). At day 26 of culture, human MGE progenitors (1 − 2 × 10⁵) were cross-linked using formaldehyde, lyzed with sodium dodecyl sulfate-based reagents, and chromatin was sonicated on a Bioruptor (Diagenode, Denville, NJ, USA) using a modified ChIP-seq protocol (43 (link)). ChIP was performed using antibodies against H3K27ac (Abcam Ab4729) and H3K27me3 (Abcam Ab4729). Prepared libraries from ChIP and input DNA were sequenced on an Illumina HiSeq instrument. For all experiments, reads were mapped to hg19 using BWA (44 (link)) and peaks were called using MACS (45 (link)). The generated ChIP-seq data will be available in a coming manuscript (Eshel et al., unpublished).

ChIP-seq is a method used to analyze protein interactions with DNA. ChIP-seq combines with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. 10⁶ differentiated cells from Day 26, Day 39, and Day 55 stages were cross-linked using 1% formaldehyde. The lysate with sodium dodecyl sulfate-based reagents and chromatin was sonicated for 18 cycles (60 s On, 60 s Off) using Bioruptor. The sonicated samples were immunoprecipitated using magnetic beads 25 μL protein A (Invitrogen cat.10002D) and 25 μL protein G (Invitrogen cat.10004D). The samples were reverse crosslinked using Proteinase K overnight at 650°C. The sonicated fragments were 300-500bp in size. The DNA fragments were purified using the phenol-chloroform protocol. ChIP was performed using antibodies against H3K27ac (Abcam Ab4729) and H3K27me3 (Abcam Ab4729). Prepared libraries from ChIP and input DNA were sequenced using the HiSeq instrument (Illumina, United States). The ChIP libraries were analyzed and mapped to hg19 using BWA (Li and Durbin, 2009 (link)) and peaks were called using MACS (Zhang et al., 2008 (link)).

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 [47 (link)] 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) [48 ]. Non-uniquely placed reads were discarded. Cisgenome v2.0 software package [49 (link)] 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.