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Sample GSM6573865 Query DataSets for GSM6573865
Status Public on Nov 21, 2022
Title CUTRUN_State1_Klf4_BR1
Sample type SRA
 
Source name State1, Klf4
Organism Mus musculus
Characteristics cell line: V6.5
cell type: embryonic stem cell line
cut&run antibody: Klf4 (Abcam ab12947)
genotype: WT
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 CUT&RUN was performed using CUTANA ChIC/CUT&Run Kit (Epicypher 14-1048) following manufacturer’s instruction in User Manual Version 2.1 or User Manual Version 3. Three technical replicates per antibody were performed. For sorted state populations, cells were harvested using versene, and 8x105 cells per replicate were isolated by flow cytometry. Harvested cells were permeabilized using 0.005% digitonin. Additionally, 1 ug of antibody per sample was added (Klf4 Abcam ab12947, Zfp281 Abcam ab10131), with Rabbit IgG antibody (CUTANA 13-0042) used as the negative control. E.coli spike-in DNA was also added to each sample.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
 
Description CUT&RUN for Klf4 in State 1
Data processing CUT&RUN reads were trimmed using cutadapt (v1.16) (-m 10 -q 10). Paired-end reads were aligned to the mm10 reference genome and the E.coli MG1655 using bowtie2 with default parameters. The normalization factor for each replicate was calculated as 1/((unique E.coli reads⁄unique mouse reads)× 100%). Normalized bigWig files were generated for each replicate using the bamCoverage function in the deeptools package by inputting the normalization factor as the --scaleFactor parameter. Replicates were further normalized to IgG negative control using the bigwigCompare function with the parameter --operation log.
Assembly: mm10
Supplementary files format and content: CUT&RUN: bigWig format
Library strategy: CUT&RUN
 
Submission date Sep 12, 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 SAMN30807455
SRA SRX17533786

Supplementary file Size Download File type/resource
GSM6573865_CUTRUN_State1_Klf4_BR1.bigWig 216.4 Mb (ftp)(http) BIGWIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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