Sample information curated by ChIP-Atlas

Antigen

Antigen Class
No description
Antigen
NA

Cell type

Cell type Class
No description
Cell type
NA
NA
NA

Attributes by original data submitter

Sample

ENA first public
2020-01-28
ENA last update
2019-02-11
ENA-CHECKLIST
ERC000011
External Id
SAMEA5332476
INSDC center alias
Department of iPS Cell Research & Epigenetic Medicine, Keio University School of Medicine
INSDC center name
Department of iPS Cell Research & Epigenetic Medicine, Keio University School of Medicine
INSDC first public
2020-01-28T04:03:21Z
INSDC last update
2019-02-11T17:11:18Z
INSDC status
public
Submitter Id
E-MTAB-7670:CTCF_noDOX_1
broker name
ArrayExpress
common name
human
genotype
STITCH/KRAB
sample name
E-MTAB-7670:CTCF_noDOX_1
sex
female

Sequenced DNA Library

library_name
CTCF_noDOX_1_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were dissociated from dish with TrypLE Select (Thermo Fisher Scientific K.K., Cat#12563-011) and washed with PBS. We cultured the cells in the StemFit® AK02N medium (ReproCELL, Cat#RCAK02N) on dish coated with iMatrix-511 (ReproCELL, Cat#NP892-012) without feeder cells. Doxycycline was added at the final concentration of 10 or 0 ng/ml to the STITCH/KRAB cells. We lysed the cells in 20 mM Tris-HCl pH8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.05% SDS, 3 mM CaCl2 and protease inhibitorsand. Then we incubated them with micrococcal nuclease (NEB, Cat#M0247S) at 37°C for 10 minutes. To stop the digestion reaction, EDTA and EGTA were added so the final concentration was 10 mM and 20 mM, respectively. To solubilize the chromatin, we applied sonication with Ultrasonic Homogenizer UH-50 (SMT Co., Ltd.) for three times of 20-seconds pulse and incubated them at 4°C for 1 hour. The solubilized chromatin after removal of the cell debris by centrifugation was incubated with antibody at 4°C for overnight. The antibodied used here were anti.H3K4me3, -H3K27me3, -H3K9me3 and -H3K27ac (MAB Institute, Cat#MABI0304S, Cat#MABI0323S, Cat#MABI0318S and Cat#MABI0309S, respectively) and anti-CTCF antibody (Millipore, Cat#07-729). The chromatin with the antibodies was incubated with 6 μl of Dynabeads Protein G (Thermo Fisher Scientific, Cat# 10003D) for one hour. Then the beads were washed extensively. The chromatin was treated with RNaseA (50 ng/μl) at 37°C for 15 minutes and then with Proteinase K (100 ng/μl) at 55°C for 1 hour in ChIP extraction buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM EDTA, 5mM EGTA, 0.1% SDS). The DNA was precipitated with ethanol and eluted in 10 mM Tris-HCl pH 8.0 after removal of the beads. To prepare nChIP-seq libraries, we used the NEBNext Ultra II DNA Library Prep with Sample Purification Beads (NEB, Cat#E7103S). We basically followed the protocol from the manufacturer, but used partly oligo DNAs that were customly designed by us for the PCR reaction.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg38

Number of total reads
5986145
Reads aligned (%)
98.0
Duplicates removed (%)
17.8
Number of peaks
12409 (qval < 1E-05)

hg19

Number of total reads
5986145
Reads aligned (%)
97.2
Duplicates removed (%)
18.4
Number of peaks
12410 (qval < 1E-05)

Base call quality data from DBCLS SRA