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Status |
Public on Nov 21, 2022 |
Title |
ChIPseq_Zfp281KO_Zfp281 |
Sample type |
SRA |
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Source name |
mouse embryonic stem cell
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Organism |
Mus musculus |
Characteristics |
tissue: mouse embryonic stem cell cell line: V6.5 cell type: embryonic stem cell line chip antibody: Zfp281 (Abcam ab10131) genotype: Zfp281 KO
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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.
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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.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina NextSeq 500 |
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Description |
ChIPseq for Zfp281KO in Zfp281KO mESC
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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
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Submission date |
May 09, 2022 |
Last update date |
Nov 29, 2022 |
Contact name |
Sofia Hu |
E-mail(s) |
sofiahu@mit.edu, xsu@cpdr.org
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Organization name |
MIT
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Street address |
500 Main Street
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City |
Cambridge |
State/province |
MA |
ZIP/Postal code |
02139 |
Country |
USA |
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Platform ID |
GPL19057 |
Series (1) |
GSE169044 |
Transcription factor antagonism regulates heterogeneity in embryonic stem cell states |
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Relations |
BioSample |
SAMN28163643 |
SRA |
SRX15213761 |
Supplementary file |
Size |
Download |
File type/resource |
GSM6124626_ChIPseq_Zfp281KO_Zfp281.bigWig |
678 b |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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