Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K4me3

Cell type

Cell type Class
Blood
Cell type
Monocytes-CD14+
Tissue
monocytes
Lineage
mesoderm
Description
Monocytes-CD14+ are CD14-positive cells from human leukapheresis production, from donor RO 01746 (draw 1 ID is RO 01746, draw 2 ID is RO 01826), newly promoted to tier 2: not in 2011 analysis

Attributes by original data submitter

Sample

source_name
CD14+ monocytes
tissue
peripheral blood
origin
dcSSc patient
chip antibody
H3K4me3 (07-473, Merck Millipore)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
PBMCs were isolated using Ficoll Paque Plus (GE Healthcare) after which CD14+ monocytes were isolated using anti-CD14 microbeads (Miltenyi Biotech, Germany) to reach >95% purity. Freshly isolated monocytes from all subjects were immediately stored for RNA or ChIP analysis.Cells used for ChIPseq or ChIP-PCR were fixed using 1% formaldehyde (Sigma-Aldrich, Saint Louis,MO, USA) for 5 min followed by the addition of 0.125M Tris (pH 7.6) to stop the crosslinking reaction. After 5 min, cells were washed three times with ice-cold PBS and lysed for 10 min in L1 buffer (50 mM Tris, pH 8.0; 2 mM EDTA; 0.1% Nonidet P-40; and 10% glycerol) supplemented with protease inhibitors (5 μg/ml leupeptin, 5 μg/ml pepstatin, and 1 mM PMSF) and phosphatase inhibitors (1 mM Na3VO4, 20 μM PAO, and 50 mM NaF). Nuclei were pelleted at 1000xg in a cold microfuge and resuspended in L2 buffer (50 mM Tris, pH 8.0; 5 mM EDTA; and 1% SDS plus inhibitors). Chromatin was then sheared by sonication (180 seconds at 1000 cycles, 17.9 of duty factor and 450 of peak power on a Covaris S220 (Woburn, MA, USA), centrifuged to pellet debris and finally 10 times diluted in dilution buffer (50 mM Tris, pH 8.0; 0.5% Nonidet P-40; 0.2 M NaCl; and 5 mM EDTA). Nuclear extracts from 1 million monocytes were immunoprecipitated with 5 μg anti-H3K4me3 (07-473, Merck Millipore, Billerica, MA, USA), anti H3K27ac (AB4729, Abcam, Cambridge, UK) DNA was purified using ChIP DNA Clean & Concentrator kit (Zymo Research), end-repair, a-tailing, and ligation of sequence adaptors was done using Truseq nano DNA sample preparation kit (Illumina). Samples were PCR amplified, checked for the proper size range and for the absence of adaptor dimers on a 2% agarose gel and barcoded libraries were sequenced 75bp single-end on Illumina NextSeq500 sequencer.

Sequencing Platform

instrument_model
NextSeq 500

hg38

Number of total reads
11159924
Reads aligned (%)
98.4
Duplicates removed (%)
21.2
Number of peaks
17227 (qval < 1E-05)

hg19

Number of total reads
11159924
Reads aligned (%)
98.2
Duplicates removed (%)
21.5
Number of peaks
17307 (qval < 1E-05)

Base call quality data from DBCLS SRA