Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Blood
Cell type
CD4 CD8 double negative cells
NA
NA

Attributes by original data submitter

Sample

source_name
Lin- BM derived DN1 cells
strain
C57BL6/J
tissue
bone marrow
cell type
DN1
treatment
Cultured on OP9DL1
chip antibody
none (input)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Lin- BM isolation / culture protocolBone marrow (BM) was removed from the femur and tibia of 2-3 month-old Cas9 mice. Suspensions of BM cells were prepared and stained for lineage markers using biotin-conjugated lineage antibodies (CD11b, CD11c, Gr1, TER-119, NK1.1, CD19, CD3ε, B220), then incubated with streptavidin-coated magnetic beads, and passed through a magnetic column. Lin− cells were eluted and cultured on OP9-DL1 monolayers using OP9 medium supplemented with 10 ng/ml of IL-7 and 10 ng/ml of Flt3L. On day 5, cultured cells were disaggregated, filtered through 40-μm nylon mesh and DN1 cells (Lin-, CD45+, Kit+,CD25-) were sorted. On day 14,DN3 ( Lin-CD45+Kit-CD25+) were harvested. Ten million of BM-derived DN1 and DN3 cells were fixed with 1 mg/ml DSG (Thermo Scientific) in PBS for 30 min at RT followed by additional 10 min with addition of formaldehyde up to 1%. The reaction was quenched by addition of 1/10 volume of 0.125M glycine and the cells were washed with HBSS (Gibco). Pelleted nucleus were dissolved in lysis buffer (0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8) and PIC) and sonicated on a Bioruptor (Diagenode) for 18 cycles of 30sec sonication followed by 30sec rest, with max power. Five μg per 107 cells of anti-Runx1 Abs (ab23980,Abcam) were hybridized to Dynabeads M-280 Sheep anti-Rabbit IgG (Invitrogen) and added to the diluted chromatin complex. They were incubated over night at 4°C, then washed and eluted for 6hr at 65°C in ChIP elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA 50 mM NaCl, 1% SDS, and 50 μg proteinase K). Precipitated chromatin fragments were cleaned up using Zymo ChIP DNA Clean & Concentrator. ChIP-seq libraries were constructed using NEBNext ChIP-Seq Library Preparation Kit (NEB #E6240) following manufacturer’s instructions. Libraries were sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt following manufacturer's instructions. Base calls were performed with RTA 1.13.48.0 followed by conversion to FASTQ with bcl2fastq 1.8.4 and produced approximately 30 million reads per sample.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

mm10

Number of total reads
31718988
Reads aligned (%)
98.2
Duplicates removed (%)
14.0
Number of peaks
406 (qval < 1E-05)

mm9

Number of total reads
31718988
Reads aligned (%)
98.0
Duplicates removed (%)
14.1
Number of peaks
472 (qval < 1E-05)

Base call quality data from DBCLS SRA