Illumina HiSeq 2500 sequencing; Chromatin immuno-precipitation and sequencing of barrelette progenitors and neurons during mouse brain development
Cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature (RT) and quenched with 125mM glycine for 5 minutes at RT. Cells were rinsed with PBS. Cells were collected by scraping. ChIP-seq library was done by ChIPmentation protocol. 1,000,000 cells (H3K4me2, H3K27ac, H3K27me3), 30,000,000 cells (Ser5P RNAPII, Ser2P RNAPII) were used. Cells were lysed in 200 - 800 μl of Sonication Buffer for 15 min on ice, and sonicated using the Covaris machine or the Bioruptor Pico machine to obtain DNA fragment the size of which distributes between 150bp and 500bp. After centrifugation, the supernatant was transferred to new tubes, diluted five times with Equilibration Buffer. Chromatin solutions were incubated over-night at 4°C with 5μg of anti-H3K4me2 (Millipore, 07-030, PRID: AB_10099880), 5μg of anti-H3K27ac (abcam, ab4729, PRID: AB_2118291), 6μg of anti-H3K27me3 (Cell Signaling, 9733, PRID: AB_2616029), 30μg of anti-Ser5P RNAPII 4H8 (Covance, MMS-128P, PRID: AB_10013820), and 30μg of anti-Ser2P RNAPII 3E10 (Active Motif, 61083, PRID: AB_2687450) antibodies. The next day, 40 - 200 μl of protein G coupled to magnetic beads were added and the incubation was continued for 2 hours at 4°C (as for anti Ser2P RNAPII 3E10 rat-derived IgG1, 200μl beads were pre-incubated with 60μl rabbit-derived anti-rat IgG antibody over-night to bridge primary antibodies and protein G). The beads were then washed twice with RIPA Buffer, and twice with 10mM Tris HCl pH8. Beads were resuspended in 30-90μl Tagmentation Buffer containing 1-3μl Tagment DNA Enzyme 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 (sample to beads ratio 1:2) 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).