Illumina HiSeq 2500 sequencing; Chromatin immuno-precipitation and sequencing of barallete progenitors and neurons during mouse brain development
Cells were dissociated by papain and cross-linked with 1% formaldehydefor 10 minutes at room temperature (RT) and quenched with 125mM glycine for 5 minutes at RT. Cells were rinsed with PBS and filtered.  6,000,000 cells were used. Cells were lysed in 200 μl of Sonication Buffer for 15 min on ice, and sonicated using the Bioruptor Pico machine to obtain DNA fragment the size of which distributes around 2Kbp. After centrifugation, the supernatant was transferred to new tubes, diluted ten times with Equilibration Buffer. Samples were pre-cleaned by protein G coupled to magnetic beads at 4°C for one hour. As for single ChIP-seq, 10% of chromatin was incubated over-night at 4°C with 1μg of anti-H3K27ac or anti-H3K27me3 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 twice times with RIPA Buffer, once with High-Salt RIPA Buffer, once with LiCl Buffer, and once with TE Buffer. DNA was eluted from the beads with 100μl Elution Buffer at 65°C for over-night. DNA was purified with MinElute PCR Purification Kit (QIAGEN, 28004). Purified large fragments (2kb) of DNA were fragmented by 1μl Tagment DNA Enzyme from the Nextera DNA Sample Prep Kit (Illumina) at 55°C for 5 minutes. After tagmentation, DNA was purified with MinElute PCR Purification Kit by 25μl 10mM Tris HCl pH8. Final enrichment of the libraries was performed in 50μl reaction. 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).