Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
IRF8

Cell type

Cell type Class
Blood
Cell type
THP-1
Primary Tissue
Blood
Tissue Diagnosis
Leukemia Acute Myelogenous

Attributes by original data submitter

Sample

source_name
THP-1 cell line
cell type
untreated monocytes
chip antibody
IRF8 (Santa Cruz sc-6058x)
cell line
THP-1

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Soluble-chromatin was prepared from 40,000,000 THP-1 cells according to previously described protocols with some modifications (outlined below). Briefly, cells were crosslinked with 1% formaldehyde (final concentration) (Fisher Scientific, cat # F79-500) for 10 minutes at room temperature with gentle shaking. Crosslinking was stopped by adding 125 mM final concentration of glycine solution in PBS . Fixed cells were pelleted at 800xg for 5 minutes at 4˚Ϲ and washed twice with 10 ml of cold PBS in a 15 ml conical tube and pelleted at 800xg for 5 minutes at 4˚Ϲ. Washed cell pellet was re-suspended in 10 ml of Lysis Buffer 133, nutated for 10 minutes at 4˚Ϲ, and pelleted at 2000xg for 5 minutes at 4˚Ϲ. The same procedure was repeated with Lysis Buffer 233 at room temperature followed by pelleting at 2000xg for 5 minutes at 4˚Ϲ. To release nuclei from hard-to-disrupt THP-1 membranes, cells were re-suspended in 10 ml of Lysis Buffer 333 and were shaken vigorously (225 rpm) at room temperature for 30 minutes. Cells then were passed through an 18-gauge needle (VWR, cat # BD305195) for 25 times using a 10ml syringe. Nuclei were pelleted at 3000xg for 20 minutes at 4˚Ϲ and re-suspended in 500 μl of Lysis Buffer 3 and then transferred into a 1.5 ml microfuge tube placed in Benchtop 1.5 ml Tube Cooler (Active Motif, cat # 53076). The nuclei were sonicated using Active Motif Q120AM sonicator with a 3.2 mm Probe (Active motif cat # 53053) at 25% amplitude for 15 minutes with 20 seconds ON and 30 seconds OFF cycles (45 cycles total). Cell-debris was pelleted at 21,000xg for 30 minutes at 4˚Ϲ. 50 μl of the combined soluble-chromatin was saved to be used as the input DNA upon reverse-crosslinking. For immunoprecipitation, 500 μl of the soluble chromatin was mixed with 30 μg of either PU.1, C/EBPα, H3K4me or H3K27ac antibodies (60 μg of IRF8 antibody was mixed with 1 ml of the soluble chromatin), and a tubes were rotated at 25 rpm for one hour at 4˚Ϲ using HulaMixer (ThermoFisher Scientific cat # 15920). 125 μl of the protein A Dynabead slurry (ThermoFisher Scientific cat # 10001D) per each rabbit antibody (PU.1. C/EBPα, H3K4me or H3K27ac), and 250 μl of the protein G Dynabead slurry (ThermoFisher Scientific cat # 10003D) for the goat-IRF8 antibody, were transferred into 1.5 ml microfuges and placed on DynaMag magnet (ThermoFisher Scientific, cat # 12321D) until all beads collected on the side of tubes. The solution was gently aspirated off from each tube and then the rack was removed from the magnet and beads were re-suspended in 1 ml of the Lysis Buffer 3 with several gentle inversions and then the rack was put back on the magnet and the buffer was aspirated off after all beads were collected on the side of the tube. Each bead then re-suspended in 50 μl of Lysis Buffer 3 and were added to the corresponding tube and returned to HulaMixer to rotate at 35 rpm for over-night at 4˚Ϲ. Dynabeads were collected with placing the tubes in the magnet. Beads were washed for 6 times with 1 ml of the Lysis Buffer 3 and two times with 1 ml of the Wash Buffer (RIPA). All ChIP samples along with the 50 μl of the soluble-chromatin were reverse-crosslinked by adding 200 μl of the Elution buffer and 3 μl of 20 mg/ml Proteinase K (ThermoFisher Scientific, cat # AM2546) and incubated at 65˚Ϲ for over-night. Beads were collected by magnets and the solutions were transferred into a new 1.5 microfuges containing 1 μl of 10 mg/ml RNase A (ThermoFisher Scientific, cat # EN0531) and left at room temperature for an hour. The ChIP and input DNA were purified using QIAquick PCR Purification Kit (QIAGEN, cat # 28104) and eluted in 50 μl of 50˚Ϲ Nuclease-Free Water (Thermo Fisher Scientific, AM9932). The concentration and size distribution of the ChIP-DNA samples were defined using Agilent 2100 Bioanalyser DNA libraries were prepared using NEBNext Ultr II DNA Library Prep kit (NEB, E7645S) following the provider's instruction manual. Amplified libraries were Bioanalyzed again to check the size selection efficiency and to define the concentrations of libraries before preparing the library pool involving the same molarity of each library

Sequencing Platform

instrument_model
Illumina HiSeq 4000

hg19

Number of total reads
46823255
Reads aligned (%)
85.7
Duplicates removed (%)
11.3
Number of peaks
7687 (qval < 1E-05)

hg38

Number of total reads
46823255
Reads aligned (%)
87.8
Duplicates removed (%)
9.9
Number of peaks
7706 (qval < 1E-05)

Base call quality data from DBCLS SRA