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Sample GSM6124623 Query DataSets for GSM6124623
Status Public on Nov 21, 2022
Title ChIPseq_Klf4KO_Klf4
Sample type SRA
 
Source name mouse embryonic stem cell
Organism Mus musculus
Characteristics tissue: mouse embryonic stem cell
cell line: V6.5
cell type: embryonic stem cell line
chip antibody: Klf4 (R&D AF3158)
genotype: Klf4 KO
Growth protocol V6.5 mouse embryonic stem cells (mESC) were cultured on tissue culture-treated 10cm plates pre-coated with 0.2% gelatin in phosphate-buffered saline (PBS). ESC were cultured in Dulbecco’s Modified Eagle Medium (Fisher), plus 10% fetal bovine serum, 1x penicillin/streptomycin, 1x non-essential amino acids, 1x β-mercaptoethanol, and 1000 U ml-1 leukemia inhibitor factor. All cells were grown at 37C and 5% CO2 and passaged every two days to maintain 10-70% confluency.
Extracted molecule genomic DNA
Extraction protocol scRNAseq: Single-cell suspension of V6.5 mESC was made by treatment with 0.25% trypsin followed by inactivation in complete medium. Single-cell isolation and library preparation was done using a 10x Genomics Chromium machine in 3’ RNA digital gene expression mode.
ChIP-seq: Cells were crosslinked in 1% formaldehyde on a rocker at room temperature for 10 minutes. Crosslinking was quenched with 250 mM glycine on a rocker at room temperature for 5 minutes. After cell lysis, chromatin was Maase-treated and sonicated using a Covaris220 evolution solicitor. ChIPseq libraries were prepared using NEB UltraII kit from 6-10 ng of DNA. Libraries were prepared by the MIT BioMicro Center.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
 
Description ChIPseq for Klf4 in Klf4KO mESC
Data processing scRNAseq: Cellranger (v.3.1.0) mkfastq and count pipelines were used for: demultiplexing, converting to fastq format, combining sequencing results, alignment to GRCm38 (mm10) genome assembly and gene annotation, barcode processing, and UMI counting.
ChIPseq: Reads were aligned to the mm10 reference genome using BWA-MEM (v.0.7.16a) with default parameters. Reads per genomic content (RPGC)-normalized bigWig files were generated for each IP using the bamCoverage function in the deeptools package (v.3.0.1) with parameters --normalizeUsing RPGC --effectiveGenomeSize 2652783500 --centerReads --extendReads 150. Background signal was subtracted from each sample ChIP using the bigwigCompare function with the parameter –operation subtract (ex. Klf4 ChIP signal in Klf4-/- cells was subtracted from Klf4 ChIP signal in WT cells and from Klf4 ChIP signal in Zfp281-/- cells).
V6.5 mouse embryonic stem cells (mESC) were cultured on tissue culture-treated 10cm plates pre-coated with 0.2% gelatin in phosphate-buffered saline (PBS+B39). ESC were cultured in Dulbecco’s Modified Eagle Medium (Fisher), plus 10% fetal bovine serum, 1x penicillin/streptomycin, 1x non-essential amino acids, 1x β-mercaptoethanol, and 1000 U ml-1 leukemia inhibitor factor. All cells were grown at 37C and 5% CO2 and passaged every two days to maintain 10-70% confluency.
Assembly: mm10
Supplementary files format and content: scRNAseq: filtered feature-barcode matrix in MEX format
Supplementary files format and content: ChIPseq: bigWig format
 
Submission date May 09, 2022
Last update date Nov 29, 2022
Contact name Sofia Hu
E-mail(s) sofiahu@mit.edu, xsu@cpdr.org
Organization name MIT
Street address 500 Main Street
City Cambridge
State/province MA
ZIP/Postal code 02139
Country USA
 
Platform ID GPL19057
Series (1)
GSE169044 Transcription factor antagonism regulates heterogeneity in embryonic stem cell states
Relations
BioSample SAMN28163640
SRA SRX15213758

Supplementary file Size Download File type/resource
GSM6124623_ChIPseq_Klf4KO_Klf4.bigWig 678 b (ftp)(http) BIGWIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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