Illumina HiSeq 2500 sequencing; Chromatin immuno-precipitation and sequencing of barallete progenitors and neurons during mouse brain development
To avoid any effects of dissociation on chromatin state, we performed ChIP-seq against H3K27ac and H3K27me3 using E14.5 PrV tissue that was pre-fixed prior to dissociation. For this, micro-dissected PrV region was directly cross-linked with 1% formaldehyde in DMEM 1X/ FCS 10% for 12 minutes at room temperature (RT) and quenched with 125mM glycine for 5 minutes at RT. The tissue was rinsed twice with DMEM 1X/ FCS 4% and once with PBS, then treated with papain digestion mix for 15 minutes at 37°C and immediately put on ice.  Tissue was rinsed by ice-cold DMEM 1X, and dissociated by pipetting and filtered. ChIP-seq library was done by ChIPmentation protocol. 500,000 were lysed in 40μl of Sonication Buffer for 15 min on ice, and sonicated using the Covaris machine to obtain DNA fragment the size of which distributes between 150bp and 500bp. The supernatant was transferred to a new tube, diluted with Equilibration Buffer. Chromatin solutions were incubated over-night at 4°C with  1μg of anti-H3K27ac (abcam, ab4729, PRID: AB_2118291) or 1μg of anti-H3K27me3 (Millipore, 07-449, PRID: AB_310624) antibodies. The next day, 20μl of protein G coupled to magnetic beads were added and the incubation was continued for 2 hours at 4°C. The beads were then washed five times with RIPA Buffer, twice with High-Salt RIPA Buffer, twice with LiCl Buffer, and twice with 10mM Tris HCl pH8. Beads were resuspended in 30μl Tagmentation Buffer containing 1μl Tagment DNA Enzyme from the Nextera DNA Sample Prep Kit (Illumina, FC-121-1030) and incubated at 37°C for 10min. The beads were washed twice with RIPA Buffer and twice with TE Buffer. DNA was eluted from the beads with 60μl Elution Buffer at 65°C for 5hours. DNA was purified with SPRI AMPure XP beads and eluted in 25μl 10mM Tris HCl pH8. Enrichment of the libraries was performed in 50μl reaction (1X KAPA HiFi Hot Start Ready Mix and 0.8μM primers). Enriched libraries were purified with size selection using SPRI AMPure XP beads (sample to beads ratio 1:0.6) to remove long fragments, recovering the remaining DNA (sample to beads ratio 1:2).