GSM3017964: H3K27ac ChIPseq KO unstimulated rep2; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Histone
Antigen
H3K27ac
Cell type
Cell type Class
Blood
Cell type
Neutrophils
MeSH Description
Immature neutrophils.
Attributes by original data submitter
Sample
source_name
HoxER-PU.1 KO neutrophils
cell type
HoxER-PU.1 KO neutrophils derived from immortalized progenitors
strain
MRP8-Cre-ires/GFP:PU.1f/f (PU.1 KO)
chip antibody
H3K27ac (abcam, ab4729)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
1.5x107 HoxER-PU.1 WT and HoxER-PU.1 KO neutrophils were harvested in a 50 ml Falcon tube. Cells were crosslinked with 1 % formaldehyde for 10 min at RT while shaking (seesaw rocker SSL4, Stuart). After crosslinking 5 ml 1 M HEPES (pH 7.5) was added and reaction was quenched by addition of 0.125 M glycine (5 min, RT, shaking). Cells were pelleted (1300 rpm, 8 min, 4°C) and washed two times with ice-cold PBS. Cell lysis was performed by swelling the pellet in ice-cold lysis buffer (10 mM Tris, pH 8.0, 10 mM NaCl, 0.2 % NP-40, 1:500 proteinase inhibitor cocktail (PIC, Sigma)) for 10 min on ice. Nuclei were pelleted by centrifugation (1800 rpm, 5 min, 4°C), and were dissolved in Non-SDS sonication buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 0.5 M EGTA, 1:500 PIC). After incubation for 10 min on ice, chromatin was transferred to TPX hard plastic reaction tubes (1.5 ml or 15 ml, Diagenode) and was sonicated for 30 minutes sonication time on high power (30 sec on, 30 sec off, 0.5 sec pulse rate) using a Bioruptor (Diagenode) and a minichiller (Huber) for cooling. After sonication, chromatin was centrifuged (13200 rpm, 15 min, 4°C) to pellet cell debris and an aliquot (10% Input) was removed and was stored at -20°C for later usage. The chromatin was diluted 10x with ChIP dilution buffer (0.01 % SDS, 1.1 % Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl) in a 2 ml reaction tube. To preclear the chromatin, 40 μl Protein A/G Agarose beads (Santa Cruz) were added, and the mixture was incubated for 30 min at 4°C under constant rotation (Rotator SB3, Stuart). Beads were pelleted (1000 rpm, 1min, 4°C) and the supernatant was transferred to a new reaction tube. 10 µg H3K4me1 (Cell Signaling, 5326) or H3K27ac antibody (abcam, ab4729) were added to the chromatin, and the mixtures were incubated overnight at 4°C under rotation. Protein A/G agarose beads (Santa Cruz) were blocked for one hour at RT in 1ml PBS containing 1.5% fish skin gelatin (Sigma). After washing, beads were resuspended in the initial volume of ChIP dilution buffer. For precipitation, 200 µl protein A/G agarose beads were added to the chromatin/antibody mix, and the solution was incubated for one hour at 4°C under rotation. The beads were pelleted (1000 rpm, 1 min, 4°C) and were washed two times in 1 ml ChIP low salt immune complex wash buffer (0.1 % SDS, 1 % Triton-X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl) while rotation for 5 min at 4°. Then, beads were washed two times in 1 ml ChIP high salt immune complex wash buffer (0.1 % SDS, 1 % Triton-X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl) and two times in 1 ml ChIP LiCl immune complex wash buffer (0.25 M LiCl, 1 % NP-40, 1 % DOC, 1 mM EDTA, 10 mM Tris, pH 8.1). Afterwards, pelleted beads were washed two times in 1 ml TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0). DNA elution was performed by addition of 250 μl ChIP elution buffer (1 % SDS, 0.1 M NaHCO3) and incubation for 15 min at RT under rotation. The beads were pelleted (1000 rpm, 1 min, 4°C) and the supernatant was transferred to a new 1.5 ml Safe-Lock tube. The beads were conducted to a second elution step, and the two eluates were combined. Protein-DNA crosslinks of samples and inputs (10 %) were reversed by addition of 20 μl 5 M NaCl (overnight, 65°C). Next day, 2 μl (20 mg/ml) proteinase K (Invitrogen), 10 µl 0.5M EDTA and 20 µl Tris-HCl pH 6.5 were added and incubated 1h at 45°C. DNA was isolated by addition of one volume phenol/chloroform/isoamyl alcohol (Roth, 25:24:1) and subsequently centrifugation of the mixture (13200 rpm, 15 minutes, 4°C). The aqueous phase containing the DNA was transferred to a new tube, and 1 µl glycogen (20 mg/ml, Sigma) was added. The precipitated DNA was resuspended in 60 µl DNase and RNase free water (Gibco) and was stored at -20°C for further applications. ChIP-Seq libraries were prepared using the NEBNext® Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) following the instructions of the supplier. The Core Facility Genomic Muenster performed library quantification by qPCR (Kapa Library Quant Kit) and single-end sequencing on an Illumina Nextseq 500.