Antigen

Antigen Class

Unclassified

Antigen

Unclassified

Cell type

Cell type Class

Blood

Cell type

Monocytes

MeSH Description

Large, phagocytic mononuclear leukocytes produced in the vertebrate BONE MARROW and released into the BLOOD; contain a large, oval or somewhat indented nucleus surrounded by voluminous cytoplasm and numerous organelles.

Sample

source_name

Human blood monocytes

tissue

Peripheral blood

treatment

cIgG priming for 24 hrs

chip_antibody

H3K4me3(#07-473, Merck Millpore)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

For ChIP-seq, purified monocytes (1×10^7 cells/mL) were pre-incubated either with cell culture medium, or cIgG (10 μg/mL) for 24 hrs. Then, cells were cross-linked with 1% formaldehyde (Sigma-Aldrich) at room temperature for 10 min. Chromatin was fragmented to 200-500 bp with Bioruptor (Diagenode; 20×30 sec cycles, low power at 4°C), cleared at 14,000 rpm at 4°C for 20 min and immunoprecipitated with 1 μg αH3K4me3 Ab (#07-473, Merck Millpore) or αH3K27ac Ab (#07-449, Merck Millpore) and 40 μL of protein A/G plus magnetic beads (Merck Millpore) per reaction at 4°C overnight. Then, the ChIPed DNA was purified with MinElute PCR purification kits (Qiagen). The quality of DNA shearing and library preparation was verified by Bioanalyzer (Agilent Tech).For RNA-seq, purified monocytes (5×10^6 cells/mL) were seeded on 60 mm dishes. Monocytes were pre-incubated either with cell culture medium (RPMI), or cIgG (10 μg/ml) for 24 hrs. In parallel, these monocytes were stimulated with LPS (10ng/ml) or not for 2 hrs. After wash-out, cells were harvested and processed into RNA extraction. Total RNA was polyA enriched, fragmented and converted into a library of Illumina-compatible sequencing templated with Illumina mRNA-seq sample preparation kit according to the manufacturer’s instructions. The cDNA library was size-fractionated in a 1% agarose gel. 200-250 bp fragments were isolated and amplified by PCR with Illumina primers PE1 and PE2 for 15 cycles. The integrity and quality of total RNA and size-selected libraries was evaluated with Bioanalyzer 2100 (Agilent Tech).

Sequencing Platform

instrument_model

Illumina HiSeq 2000

hg38

Number of total reads

24042364

Reads aligned (%)

98.5

Duplicates removed (%)

9.5

Number of peaks

18726 (qval < 1E-05)

hg19

Number of total reads

24042364

Reads aligned (%)

97.8

Duplicates removed (%)

10.7

Number of peaks

18740 (qval < 1E-05)