Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K4me1

Cell type

Cell type Class
Uterus
Cell type
JHUEM-14
Primary Tissue
Uterus
Tissue Diagnosis
Adenocarcinoma Endometrioid

Attributes by original data submitter

Sample

source_name
JHUEM14_Veh
cell line
JHUEM-14
cell type
endometrial cancer cell line
treatment
10nM DMSO
chip antibody
H3K4Me1 (Abcam #ab8895)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were washed twice with PBS and fixed at room temperature in 1% formaldehyde in PBS. After 10 min, cells were placed on ice and washed twice with ice-cold PBS. The reaction was quenched with 10 mM DTT in 100 mM Tris.HCl (pH 9.4) and cells removed from the dish with a cell scraper. Cells were incubated at 30oC for 15 min then spun for 5 min at 2,000 g. Cells were washed sequentially with ice cold PBS, Buffer I (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 6.5) and Buffer II (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH6.5) and centrifuged for 5 min at 2,000 g at 4 oC. Cells were resuspended in 300-750 ul of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris.HCl, pH 8.1, with complete protease inhibitor cocktail (Sigma Aldrich #11836145001). Ishikawa cells were sonicated for eight cycles (10s) and JHUEM-14 cells for 20 cycles using the highest power setting of a Branson Digital Sonifier SLPt. After chromatin shearing was confirmed by agarose gel electrophoresis, samples were centrifuged for 10 min at 4 oC. Samples were diluted 10-fold in 1% Triton X-100, 2 mM EDTA, 20 mM Tris.HCl (pH 8.1) and 150 mM NaCl with complete protease inhibitor cocktail. Magna ChIP protein A/G magnetic beads (EMD Millipore #16-663) were added to 500 ul of diluted chromatin and incubated with 5 ug of antibody overnight at 4 oC. Antibodies to H3K4Me1 (Abcam #ab8895), H3K4Me3 (Abcam #ab8580) and H3K27Ac (Abcam #ab4729) were used. The next day supernatant was removed and the beads washed three times with the following ice-cold buffers: RIPA 150 (0.1% SDS, 1% Triton X-100, 1 mM EDTA, 50 mM Tris.HCl (pH 8.10, 150 mM NaC1, 0.1% sodium deoxycholate), RIPA 500 (0.1% SDS, 1% Triton X-100, 1 mM EDTA, 50 mM Tris.HCl (pH 8.10, 500 mM NaC1, 0.1% sodium deoxycholate), LiCl RIPA (500 mM LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 50 mM Tris.HCl (pH 8.1) and TE buffer. Chromatin was then eluted by incubating beads overnight at 60 oC with 100 ul of elution buffer (1% SDS, 100 mM NaHCO3) and 0.5 mg/ml proteinase K. The next day beads were incubated at 95 oC for 10 min and supernatant removed. Chromatin was purified using the QIAquick Spin kit (QIAGEN) and eluted from columns using 50 ul of QIAGEN EB buffer. DNA was quantified using a Qubit dsDNA HS Assay kit (ThermoFisher Scientific). Libraries were prepared by the Australian Genomic Research Facility for library preparation and sequencing on the Illumina HiSeq 2500

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg38

Number of total reads
69509975
Reads aligned (%)
96.3
Duplicates removed (%)
30.7
Number of peaks
3034 (qval < 1E-05)

hg19

Number of total reads
69509975
Reads aligned (%)
94.1
Duplicates removed (%)
35.4
Number of peaks
2206 (qval < 1E-05)

Base call quality data from DBCLS SRA