Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Blood
Cell type
Bone Marrow Cells
MeSH Description
Cells contained in the bone marrow including fat cells (see ADIPOCYTES); STROMAL CELLS; MEGAKARYOCYTES; and the immediate precursors of most blood cells.

Attributes by original data submitter

Sample

source_name
c-Kit+ Bone Marrow cells
antibody
none
genotype/variation
Asxl1_KI

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Hematopoietic cells were harvested from long bones that were triturated and passed through 40-μm nylon mesh to obtain a single cell suspension. Isolation of c-Kit+ BM cells was performed with anti-CD117 MicroBeads, and separated on a MACS Column in the magnetic field of a MACS Separator (Miltenyi Biotec).  RNA-seq: Library preparation for whole transcriptome analysis was performed according to the manufacturer's instructions (SureSelect Strand Specific RNA Preparation Kit; Agilent Technologies, Santa Clara, CA, USA). Briefly, poly(A) RNA purified from 2 μg of total RNA was chemically fragmented to appropriate sizes. First-strand cDNA was synthesized from the fragmented poly(A)-selected mRNA, followed by the synthesis of second-strand cDNA. Then, adaptor oligo-DNA was ligated to both ends of the double-stranded cDNA. Libraries were prepared with 12 cycles of PCR amplification of adaptor-ligated cDNA using primers that are complimentary to the adaptors. The libraries were sequenced by GAIIx next-generation sequencer (Illumina, San Diego, CA, USA) using the single-end 36 bp sequencing protocol. The generated sequence tags were mapped onto the murine genomic sequence (mm10; UCSC Genome Browser) and mRNA expression levels were normalized as reads per kilobase per million.  ChIP-seq : For ChIP-seq in murine cells 10 million BM c-Kit+ cells, isolated with anti-mouse c-Kit Biotin (13-1171-85, eBioscience) and Anti-Biotin Microbeads (130-090-485, Miltenyi Biotec), were used. Briefly, cells were fixed in a 1% methanol-free formaldehyde solution and then resuspended in sodium dodecyl sulfate (SDS) lysis buffer. Lysates were sonicated in an E220 focused-ultrasonicator (Covaris) to a desired fragment size distribution of 100–500 base pairs. IP reactions were performed with the following antibodies, each on approximately 500,000 cells as previously described (Krivtsov et al., 2008); Antibodies used for ChIP include anti-H3K4me3 (Cell Signaling 9751S), anti-H3K27me3 (Millipore 07-449), anti-H2AK119Ub (Cell Signaling 8240), anti-Asxl1 (Santa Cruz Biotechnology, sc-85283), and anti-FLAG (Sigma-Aldrich, F1804).  ChIP assays were processed and barcoded library and raw data analysis were performed as previously described (Micol et al., 2017). 

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
31714851
Reads aligned (%)
97.8
Duplicates removed (%)
6.8
Number of peaks
433 (qval < 1E-05)

mm9

Number of total reads
31714851
Reads aligned (%)
97.7
Duplicates removed (%)
6.9
Number of peaks
401 (qval < 1E-05)

Base call quality data from DBCLS SRA