Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K4me2

Cell type

Cell type Class
Breast
Cell type
SUM 159PT
Primary Tissue
Breast
Tissue Diagnosis
Anaplastic Carcinoma

Attributes by original data submitter

Sample

source_name
breast cancer cells
cell line
SUM159
antibody
anti-H3K4me2 (Millipore, 07-030)
treatment
C70
time
2day

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For KDM5B ChIP-seq, 1 × 10^7 cells were fixed with 2mM DSG (Thermo scientific 20593) for 30 min at room temperature. DSG was then removed and replaced with fixing buffer (50 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 1mM EDTA) containing 1% paraformaldehyde (Electron Microscopy Sciences, 15714) and crosslinked for 10 min at 37 °C. For histone modification ChIP-seq, 5 × 10^6 cells were fixed with 1% paraformaldehyde for 10 min at room temperature. For ER ChIP-seq, 1 × 10^7 cells were fixed with 1% paraformaldehyde for 10 min at 37 °C. Crosslinking was quenched by adding glycine to a final concentration of 0.125 M. The cells were washed with ice-cold PBS, harvested in PBS. The nuclear fraction was extracted by first resuspending the pellet in 1 ml of lysis buffer (50 mM HEPES-NaOH (pH 8.0), 140 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100) for 10 min at 4 °C. Cells were pelleted, and washed in 1 ml of wash buffer (10 mM Tris-HCL (pH 8.0), 200 mM NaCl, 1 mM EDTA) for 10 min at 4 °C. Cells were then pelleted and resuspended in 1 ml of shearing buffer (10 mM Tris-HCl (pH 8), 1 mM EDTA, 0.1% SDS) and sonicated in a Covaris sonicator. Lysate was centrifuged for 5 min at 14,000 rpm to purify the debris. Then 100 µl of 10% Triton X-100 and 30 µl of 5M NaCl were added. The sample was then incubated with 20 µl of Dynabeads Protein G (LifeTechnologies,10003D) for 1 h at 4 °C. Primary antibodies were added to each tube and immunoprecipitation (IP) was conducted overnight in the cold room. Cross-linked complexes were precipitated with Dynabeads Protein G for 2 hr at 4 °C. The beads were then washed in low salt wash buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 10 mM EDTA, and 1% SDS) for 5 min at 4 °C, high salt wash buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS) for 5 min at 4 °C and LiCl wash buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS) for 5 min at 4 °C. DNA was eluted in elution buffer (100 mM sodium bicarbonate and 1% SDS). Cross-links were reversed overnight at 65 °C. RNA and protein were digested with 0.2 mg ml−1 RNase A for 30 min at 37 °C followed by 0.2 mg ml−1 Proteinase K for 1 h at 55 °C. DNA was purified with phenol-chloroform extraction and isopropanol precipitation. ChIP-seq libraries were prepared using the Rubicon ThruPLEX DNA-seq Kit from 1 ng of purified ChIP DNA or input DNA according to the manufacturer's protocol.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg38

Number of total reads
56552152
Reads aligned (%)
96.4
Duplicates removed (%)
49.1
Number of peaks
29058 (qval < 1E-05)

hg19

Number of total reads
56552152
Reads aligned (%)
95.8
Duplicates removed (%)
50.7
Number of peaks
28809 (qval < 1E-05)

Base call quality data from DBCLS SRA