Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K27me3

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++ CD16- monocytes from blood donor 4
cell type
CD14++ CD16- monocytes
chip antibody
H3K27me3 Abcam ab6002
donor id
P4
disease state
control participant

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
A blood sample of 30 ml was taken and peripheral blood mononuclear cells were isolated using ficoll-based density gradient centrifugation. Further isolation of CD14++ CD16- monocytes was conducted using magnetic cell sorting to deplete CD16++ monocytes and separate CD14++ monocytes. Purity of isolation was checked by flow cytometric analysis of the CD14+ cell fraction. Cells were cross-linked for 10 min using formaldehyde (final concentration 1%). Fixation was quenched by addition of glycine to a final concentration of 125 mM. After washing, cells were resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris/HCl, pH8.1, 1x concentrated protease inhibitors) and sonciated using a Branson-250 Sonicator (http.www.bransonultrasonics.com) using the microtip setting. Fragment size of the obtained chromatin was checked to be between 100 bp and 300 bp. Sheared chromatin was diluted 1/10 with dilution buffer (SDS 0.01%, Triton X-100 1%, EDTA 1.2 mM, Tris/HCl, pH 8.1 16.7 mM, NaCl 167 mM). Chromatin was precleared using a protein A/G-sepharose mixture for 1 hr at 4°C and incubated with apprpriate antibodies over night. Immune complexes were precipitated with protein A/G-sepharose and washed with low salt (SDS 0.1%, Triton X-100 1%, EDTA 2 mM, Tris/HCl, pH8.1 20 mM, NaCl 150 mM), high salt (SDS 0.1%, Triton X-100 1%, EDTA 2 mM, Tris/HCl, pH8.1 20 mM, NaCl 500 mM), LiCl buffer (LiCl 0.25 M, NP40 1%, Deoxycholat 1%, EDTA 1 mM, Tris/HCl, pH8.1 10 mM) and twice with TE. After crosslink reversal, immunprecipitated nucleic acids were purified on GFX columns (GE Healthcare). ChIP-seq library preparation using MicroPlex kit from Diagenode

Sequencing Platform

instrument_model
Illumina HiSeq 1000

hg38

Number of total reads
54712013
Reads aligned (%)
97.4
Duplicates removed (%)
76.2
Number of peaks
1188 (qval < 1E-05)

hg19

Number of total reads
54712013
Reads aligned (%)
96.5
Duplicates removed (%)
78.1
Number of peaks
1136 (qval < 1E-05)

Base call quality data from DBCLS SRA