Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K4me3

Cell type

Cell type Class
Blood
Cell type
Macrophages
MeSH Description
The relatively long-lived phagocytic cell of mammalian tissues that are derived from blood MONOCYTES. Main types are PERITONEAL MACROPHAGES; ALVEOLAR MACROPHAGES; HISTIOCYTES; KUPFFER CELLS of the liver; and OSTEOCLASTS. They may further differentiate within chronic inflammatory lesions to EPITHELIOID CELLS or may fuse to form FOREIGN BODY GIANT CELLS or LANGHANS GIANT CELLS. (from The Dictionary of Cell Biology, Lackie and Dow, 3rd ed.)

Attributes by original data submitter

Sample

source_name
Macrophages, exposed to RPMI media for 6 days
biomaterial_type
Primary Cell
cell_line
-
study_name
BLUEPRINT ChIP-seq data for cells in the haematopoietic lineages, from adult and cord blood samples.
center_name
NCMLS
donor_id
Healthy_donor_1
cell_type
Monocyte
tissue_type
Adult Human Peripheral Blood
disease
None
experiment_type
H3K4me3
biomaterial_provider
Sanquin Blood bank, Nijmegen, the Netherlands
treatment
Macrophages, exposed to RPMI media for 6 days
chip antibody
Rabbit polyclonal anti-H3K4me3 (Diagenode, cat. # pAb-003-050)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Purified cells were fixed with 1% formaldehyde (Sigma) at a concentration of approximately 10 million cells/ml. Fixed cell preparations were sonicated using a Diagenode Bioruptor UCD-300 for 3x 10 minutes (30 seconds on; 30 seconds off). 67µl of chromatin (1 million cells) was incubated with 229µl dilution buffer, 3µl protease inhibitor cocktail and 0.5-1µg of H3K27ac or H3K4me3 antibodies (Diagenode) and incubated overnight at 4ºC with rotation. Protein A/G magnetic beads were washed in dilution buffer with 0.15% SDS and 0.1% BSA, added to the chromatin/antibody mix and rotated for 60 minutes at 4°C. Beads were washed with 400µl buffer for 5 minutes at 4°C with five rounds of washes. After washing chromatin was eluted using elution buffer for 20 minutes. Supernatant was collected, 8µl 5M NaCl, 3µl proteinase K were added and samples were incubated for 4 hours at 65°C.Finally samples were purified using Qiagen; Qiaquick MinElute PCR purification Kit and eluted in 20µl EB. Illumina library preparation was done using the Kapa Hyper Prep Kit. For end repair and A-tailing double stranded DNA was incubated with end repair and A-tailing buffer and enzyme and incubated first for 30 minutes at 20°C and then for 30 minutes at 65°C.Subsequently adapters were  ligated  by adding 30µl ligation buffer, 10 Kapa l DNA  ligase, 5µl diluted adaptor  in a total volume of 110µl and incubated for 15 minutes at 15°C.Post-ligation cleanup was performed using Agencourt AMPure XP reagent and products were eluted in 20µl elution buffer. Libraries were amplified by adding 25µl 2x KAPA HiFi Hotstart ReadyMix and 5µl 10x Library Amplification Primer Mix and PCR, 10 cycles. Samples were purified using the QIAquick MinElute PCR purification kit and 300bp fragments selected using E-gel. Correct size selection was confirmed by BioAnalyzer analysis.   Sequencing was performed using Illumina HiSeq 2000 machines and generated 43bp single end reads.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg38

Number of total reads
33602998
Reads aligned (%)
95.4
Duplicates removed (%)
15.7
Number of peaks
27882 (qval < 1E-05)

hg19

Number of total reads
33602998
Reads aligned (%)
95.1
Duplicates removed (%)
16.1
Number of peaks
27865 (qval < 1E-05)

Base call quality data from DBCLS SRA