Sample information curated by ChIP-Atlas

Antigen

Antigen Class
No description
Antigen
NA

Cell type

Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA

Attributes by original data submitter

Sample

ENA first public
2019-02-17
ENA last update
2018-07-16
ENA-CHECKLIST
ERC000011
External Id
SAMEA4791349
INSDC center alias
Philipps-University Marburg, Genomics Core Facility
INSDC center name
Philipps-University Marburg, Genomics Core Facility
INSDC first public
2019-02-17T17:02:49Z
INSDC last update
2018-07-16T14:36:06Z
INSDC status
public
Submitter Id
E-MTAB-7011:E14.S-KD.wt_KAP1
broker name
ArrayExpress
cell line
ES-E14
cell type
embryonic stem cell
common name
house mouse
disease
normal
genotype
shSMARCAD1; SMARCAD1-3xFLAG
organism part
inner cell mass
phenotype
SMARCAD1-3xFLAG
sample name
E-MTAB-7011:E14.S-KD.wt_KAP1

Sequenced DNA Library

library_name
E14.S-KD.wt_KAP1_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
KAP1 antibody, ab22553 (abcam), was used in inducible SMARCAD1 knockdown cells prior to and after two day induction of SMARCAD1 knockdown as well as in SMARCAD1 knockdown cells that express a FLAG tagged SMARCAD1 transgene; either wild-type or an ATPase mutant. FLAG antibody used was F1804 (Sigma) in SMARCAD1 knockdown cells expressing no FLAG tagged protein (negative control) and in cells expressing FLAG tagged SMARCAD1; either wild-type or an ATPase mutant. Mouse E14 ES cells were cultivated feeder-cell free on gelatin-coated dishes in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies) supplemented with 15% foetal calf serum, 2 mM L-glutamine, 1x non-essential amino acids, 50 μM 2-mercaptoethanol, penicillin/streptomycin and 1000U/ml ESGRO leukemia inhibitory factor. To generate an enzymatically inactive SMARCAD1 chromatin remodeler a mutation in the ATP binding pocket (K523R) was generated by site-directed mutagenesis using the QuikChange Lighting Site Directed Mutagenesis Kit (Agilent 210518) and the following oligos: Fwd 5'-GCAGACGAAATGGGCCTAGGAAGAACCATTCAAGCCATTGC -3' Rev 5'- GCAATGGCTTGAATGGTTCTTCCTAGGCCCATTTCGTCTGC-3'. A Triple-FLAG tagged SMARCAD1 wild-type (E14 S-KD.WT) and ATPase mutant (E14 S-KD.ATPase_mt) constructs were stably integrated into E14 cells carrying a doxycycline inducible shRNA (E14.) targeting the 3'UTR of SMARCAD1 (shSMARCAD1) as described (Ding et al., 2017) prior to knocking down the endogenous SMARCAD1 (E14 S-KD). Doxycycline (0.5µg/ml) treatment was performed for 2 days to achieve knockdown of endogenous SMARCAD1 before ChIP was carried out. E14 chromatin cross-linking was performed with 2 mM DSG (disuccinimidyl glutarate, Thermo Scientific) in PBS for 45 min at room temperature, washed three times with PBS, and formaldehyde (Polysciences) was added to a final concentration of 1 % for 10 min at room temperature. Formaldehyde was quenched using glycine (0.125 M). Chromatin was eluted in 1% SDS, 100mM NaHCO3 and DNA purified by Qiaquick PCR cleanup columns (Qiagen). Six (FLAG) or four (KAP1) individual ChIPs were pooled and purifed on QIAquick columns (Qiagen). Five nanograms of precipitated DNA were used for library preparation using the Microplex library preparation kit v2 (Diagenode). Libraries were purified on AMPure magnetic beads (Beckman Coulter).

Sequencing Platform

instrument_model
Illumina HiSeq 1500

mm10

Number of total reads
29416378
Reads aligned (%)
97.1
Duplicates removed (%)
14.9
Number of peaks
643 (qval < 1E-05)

mm9

Number of total reads
29416378
Reads aligned (%)
96.8
Duplicates removed (%)
14.9
Number of peaks
669 (qval < 1E-05)

Base call quality data from DBCLS SRA