GSM6519849: NPCs D868N ChIP Input; Homo sapiens; ChIP-Seq
To perform ChIP experiment 200000 cells for each experimental replicate were used. Cells were cross-linked in 1% formaldehyde for 10' minutes at room temperature.  Cells were lysed in ChIP-lysis Buffer (10mM Tris-HCl 8.0,1% SDS, 5 mM EDTA). Sonication was performed using a Diagenode Bioruptor Pico sonicator (65 cycles, 30” ON-30” OFF, each cycle). Input were collected before precipitation. Samples (two technical replicates for each ChIP) were then incubated overnight at 4° using primary antibodies for H3K27ac, H3K27me3, H3K9me3 and SET in iC buffer from iDeal ChIP‐seq kit for Histones (Diagenode, cod. C01010051).   The next day, incubation with magnetic beads was performed on a rotator wheel for 3 hours at 4°. Beads were subsequently washed using iW1, iW2,iW3 and IW4 buffer (Diagenode). ChIP-DNA was then eluted, decrosslinked for 30' at 37° and 4 hours at 65° and purified using.  After quantification 5 ng of DNA were used to build libraries using NebNext-UltraII kit (NEB, cod.). Samples were sequenced paired-end NovaSeq6000 by GeneWiz. TruSeq