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Sample GSM3983258 Query DataSets for GSM3983258
Status Public on Oct 21, 2019
Title B6TS_input_b_Rep2
Sample type SRA
 
Source name TS cell line
Organism Mus musculus
Characteristics strain: C57BL/6
cell type: TS cell line
chip antibody: none
Growth protocol Mouse ES cells were cultured on feeder cells in knockout DMEM (Life Technologies) containing recombinant human leukemia inhibitory factor culture supernatant for mouse ES cell culture, GlutaMAX, 2-mercaptoethanol, non-essential amino acids, and 15% KSR (all from Life Technologies). Mouse TS cells were cultured on feeder cells in the presence FGF-4 (PeproTech, London, UK) and heparin (Sigma Chemical Company) to maintain their undifferentiated state.
Extracted molecule genomic DNA
Extraction protocol Trypsinised feeder-free ES and TS cells (1×10^6) were collected and fixed with 1% formaldehyde at room temperature. Crosslinking reaction was quenched with 125 mM glycine for 5 min. The cells were resuspended in SDS lysis buffer (ChIP Reagent, Nippon Gene Co., Ltd.), and the lysate was sonicated to fragment chromatin using a Covaris S220 (Covaris, Woburn, MA, USA). The sonicated lysate was subjected to immunoprecipitation with a monoclonal antibody against TEDA4, KLF5, H3K4me1, or H3K27ac.
The immunoprecipitated and reverse-crosslinked DNA was purified using AMPure XP beads (Beckman Coulter, Inc., Pasadena, CA, USA) according to the manufacturer’s instructions. ChIP or input DNA (0.1 ~ 1 ng) was subjected to end repair, dA-tailing, and adaptor-ligation, and amplified by 6-13 cycles of PCR.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2500
 
Data processing Single end sequencing (51bp or 101bp) was performed on the HiSeq2500 system (illumina).
Basecalls were performed using the Real-Time Analysis (RTA) version 1.12.4.2 and CASAVA_v1.8.1.
For ChIP-seq data for TEAD4 and KLF5, single-end reads (51 bp) were mapped to the mm9 reference genome using the COBWeB Algorithm implemented in Avadis NGS (Agilent). Reads with average base quality below 30, PCR duplicate reads, non-primary multiply mapped reads were removed. The resultant mapped reads (.bam file) were subjected to the MACS peak detection algorithm implemented in Strand NGS (Agilent) to detect ChIP-seq peaks with the mapped reads from the matching IgG library as a control.
For ChIP-seq data for histone modifications, single-end reads (51 or 101 bp) from each sample were first trimmed by removing adapters and low quality bases at ends using Trimmomatic 0.22, and were aligned to the mouse reference genome (mm9) using the Burrows-Wheeler Aligner 0.6.2. Uniquely mapped reads were selected by a custom script, converted from sam to bam using SAMtools 0.1.18. Reads with mapping quality < 20 were removed using SAMtools 0.1.18. The uniquely mapped reads (.bam files) were subjected the MACS peak detection algorithm (version 1.4.2 to identify ChIP-seq peaks with the matched input DNA reads as a control.
Genome_build: mm9
Supplementary_files_format_and_content: MACS peak calling output
 
Submission date Jul 28, 2019
Last update date Oct 21, 2019
Contact name Kazuhiko Nakabayashi
E-mail(s) nakabaya-k@ncchd.go.jp
Organization name National Research Institute for Child Health and Development
Department Department of Maternal-Fetal Biology
Street address 2-10-1 Okura
City Setagaya
State/province Tokyo
ZIP/Postal code 157-8535
Country Japan
 
Platform ID GPL17021
Series (2)
GSE109250 Inter-chromosomal enhancer-promoter interaction critical for the appropriate level of Tead4 gene expression at the blastocyst stage [ChIP-Seq]
GSE109252 Inter-chromosomal enhancer-promoter interaction critical for the appropriate level of Tead4 gene expression at the blastocyst stage
Relations
BioSample SAMN12388200
SRA SRX6605295

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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