GSM4232324: CD34_KM3_Replicate2_H3K4me3; Homo sapiens; ChIP-Seq
[ChIP-seq]  After crosslinking cells by using formaldehyde 1% (final concentration) for 7 minutes at room temperature, glycine 0.125nM (final concentration) was added for 5 minutes at room temperature to stop the reaction. Fixed cells were washed with PBS, resuspended in 1mL of a first lysis buffer (Triton 0.25%, Tris pH 8.0 10mM, EDTA 10mM, EGTA 0.5mM, protease inhibitor 1X) for 5 minutes, and in 1 mL of a second buffer for 30 minutes (NaCl 200mM, Tris pH 8.0 10mM, EDTA 1mM, EGTA 0.5mM, protease inhibitor 1X). Lysate was resuspended in another buffer (0.5% SDS, 0.5% Triton, Tris pH 8.0 10mM, NaCl 140mM, EDTA 1mM, EGTA 0.5mM, protease inhibitor 1X) and then sonicated by using a Covaris S2 sonicator. The parameters used were as follows: duty Factor 10%, 200 cycles per burst, 105W, 4 minutes. The supernatant collected after the spinning at 13000 rpm for 5 minutes was used for future experiments.  Beads (Protein G Dynabeads™ ; resuspension buffer : Triton 1%, Tris pH 8.0 10mM, NaCl 150mM, EDTA 2mM) were incubated overnight on a rotator at 4°C with 3μg of antibodies recognizing histone marks (H3 : #ab1791, H3K4me3 : #ab8580-100, H3K27me3 : Millipore 07-449, H3K79me2 : #ab3594, H3K27ac : Millipore 07-360). Sonicated chromatin was incubated with 250μL of beads during 4 hours on a rotator at 4°C. The beads were washed sequentially with different buffers: 'Low Salt' Buffer (0.5% NP40, 15mM KCl, 10mM Tris pH 8.0, 1mM EDTA), 3 different 'High Salt' buffers (0.5% Triton, 10mM Tris pH 8.0, 100mM (2) or 400mM (3) or 500mM (4) NaCl), twice in LiCl Buffer (0.5% NP40, 250mM LiCl, 10mM Tris pH 8.0, 1mM EDTA). ChIPed material was then eluted a classic elution buffer (10mM Tris pH8.0, 1mM EDTA) and incubated overnight at 65°C with shaking (1200rpm). An RNase and proteinase K digestion was performed in order to reverse crosslink. DNA was extracted and purified by a classic phenol-chloroform protocol. [ATAC-seq]  ATAC-seq experiment was based on method published by Buenrostro et al. (PMID: 24097267) TruSeq ChIP Library Preparation Kit by Illumina (#IP-202-9001) was used to construct DNA libraries as described in the manufacturer's instructions with a PCR for a total of 12 cycles using the Illumina indexed library primers. NextSeq 500 was used to sequence samples (75pb paired-end) according to standard Illumina protocols.
GEO Accession
GSM4232324