GSM3017969: JUNB ChIPseq unstimulated KO rep2; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
Junb
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
JUNB Cell Signaling (3753S)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
1-2x107 differentiated HoxER-PU.1 WT or HoxER-PU.1 KO neutrophils were harvested in a 50 ml Falcon tube and crosslinking was performed by adding 1 % formaldehyde followed by 10 min incubation at RT on a seesaw rocker SSL4 (Stuart). Fixation reaction was quenched by addition of glycine at a final concentration of 0.15 M and incubation for 5 min at RT while shaking. Cells were pelleted by centrifugation (290 g, 5 min, 4°C) and washed two times with ice-cold PBS. Cell pellets were resuspended in 1 ml ice-cold PBS, and cell suspensions were transferred to a 1.5 ml reaction tube. Cells were pelleted by centrifugation (850 g; 5 min, 4°C), were snap frozen in liquid nitrogen and stored at -80°C. Cell pellets were thawed on ice, were resuspended in 300 µl RIPA buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 140 mM NaCl, 1 % (v/v) Triton X-100, 0.1% (v/v) SDS, 0.1% (w/v) Na-DOC) and lysed for 10 min on ice. Cell lysates were transferred to 1.5 ml TPX hard plastic reaction tubes (Diagenode) and were sonicated using a Bioruptor (Diagenode). Sonication was performed for 40 minutes on high power settings with 15 sec on and 30 sec off, and cooling system was set to 4°C. Sheared chromatin was centrifuged (20000 g, 10 min, 4°C) and transferred to new reaction tubes. 15 µg of JUNB antibody (Cell Signaling, 3753S) or normal rabbit IgG (abcam, ab171870) were coupled to washed 75 µl Protein G Dynabeads (Thermo Fisher Scientific), then 300 µl binding/blocking buffer (0.5 % (w/v) BSA, 0.5 % (v/v) Tween-20 in PBS) was added, and the mixture was incubated for one hour at RT under rotation (Rotator SB3, Stuart). Afterwards, beads were washed once in 300 µl binding/blocking buffer, and 300 µl of chromatin was added directly to the beads. The mixtures were incubated overnight at 4°C under rotation. Beads were magnetically separated, resuspended in 180 µl ice-cold RIPA buffer and transferred to a well of a 96-well Twin tec microbiology PCR plate (Eppendorf). Beads were washed five times in ice-cold RIPA buffer, followed by two washing steps with ice-cold RIPA-500 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 500 mM NaCl, 1 % (v/v) Triton X-100, 0.1% (v/v) SDS, 0.1% (w/v) Na-DOC) and LiCl buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 250 mM LiCl, 0.5 % (v/v) NP-40, 0.5 % (w/v) Na-DOC). After a final wash step with ice-cold TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0), ChIP DNA was eluted from the beads by adding 50 µl direct elution buffer (10 mM Tris-HCl, pH 8.0, 5 mM EDTA, pH 8.0, 300 mM NaCl, 0.5 % (v/v) SDS). RNase treatment was performed by incubation ChIP DNA with 2 µl RNase (Roche) for 30 min at 37°C. Next, 2.5 µl Proteinase K (Invitrogen) and 1 µl glycogen (Roche) were added, and the mixture was incubated for 2 h at 37°C. Decrosslinking was performed by incubating the mixture at 65°C overnight. After decrosslinking magnetic beads were discarded and the supernatant was transferred to a new well of a 96-well plate. ChIP DNA was purified by adding first the 0.4x volume of Agencourt AMPure XP beads (Beckman Coulter Genomics) to bind large DNA fragments. The supernatant was then transferred to a new well of 96-well plate. Then 1.2x volume of Agencourt AMPure XP beads were added, and the beads were washed two times with 80% ethanol. ChIP DNA was eluted from the beads by 35 µl 10 mM Tris-HCl (pH 8.0), and the DNA was transferred to a new well of 96 well plate. Plates were stored at -20°C until libraries were prepared. 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.