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Sample GSM6437125 Query DataSets for GSM6437125
Status Public on Jan 18, 2023
Title FL_wt_eklf_input_rep2
Sample type SRA
Source name embryonic day E13.5 fetal liver
Organism Mus musculus
Characteristics tissue: embryonic day E13.5 fetal liver
cell line: Klf1 / Klf1-Nan
cell type: primary erythroid
genotype: Nan strain WT
chip antibody: none
Extracted molecule genomic DNA
Extraction protocol Fetal livers were dissected from E13.5 mouse embryos and mechanically dispersed into single cells by gentle pipetting and straining through a 70µM filter into ice-cold PBS containing 2% FBS. Primary fetal liver cells were crosslinked in 1% formaldehyde and cell pellets were stored at -80ºC. Frozen cells were thawed and resuspended in MNase lysis buffer (0.2% NP-40, 10mM NaCl, 10mM Tris pH 8.0) and kept on ice for 15 mins. Cells were pelleted and resuspended in MNase lysis buffer with 1mM CaCl2, 40 units Micrococcal nuclease (MNase) and incubated at 37ºC for 15 minutes. The MNase reaction was stopped by adding EDTA to a final concentration of 2mM and nuclei were pelleted by centrifugation. The pellet was resuspended in 1X RIPA buffer with 1X Protease Inhibitor Cocktail and sonicated using a cup-horn sonicator (Ultrasonic VC505) at 80 Amplitude for 50 cycles with pulses of 40s on and 50s off. The lysed and fragmented chromatin was cleared by centrifugation and 5%v/v was stored at -80ºC as input. 5-10µg of antibody (depending on the titer) was added to the lysate along with a control IgG (for enrichment estimation) and antibody-lysate mixture was incubated at 4ºC for 16h (overnight). The lysate-antibody mixture was then incubated with Protein-A or Protein-G Dynabeads, depending on the antibody serotype, for 2h at 4ºC. Immunoprecipitated complexes on Dynabeads were then washed three times in 1X RIPA with protease inhibitors, and three times in wash buffer containing 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris pH 8.0, 500mM NaCl, and protease inhibitors. This was followed by three washes in wash buffer containing 1% NP-40, 1% sodium deoxycholate, 1mM EDTA, 10mM Tris pH 8.0, and 250 mM LiCl. Finally, beads were washed three times in 1X TE buffer containing 10mM Tris pH 8.0 and 1mM EDTA. Elution was performed by adding elution buffer containing 10mM Tris pH 8.0, 1mM EDTA, 200mM NaCl and 1% SDS and incubating at 65ºC in a Thermomixer (Eppendorf Cat# 5382000023) with gentle mixing for 20 mins. Eluate containing ChIP DNA and input chromatin was removed and incubated at 65ºC with gentle mixing overnight for reverse crosslinking. DNA was treated with 0.1µg/µl Proteinase K for 1h at 55ºC followed by purification using Phenol:chloroform extraction and ethanol precipitation. ATAC-Seq was performed exactly as in (Buenrostro et al., 2013; Buenrostro et al., 2015).
ChIP-Seq libraries were made using a Neb Next DNA Ultra II DNA library preparation kit following manufacturer’s instructions. Libraries were quantified using Qubit fluorometer (Invitrogen) and library quality was assessed using a high-sensitivity DNA kit on Agilent Bioanalyzer, followed by Illumina next generation sequencing with single-end 100bp reads
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NovaSeq 6000
Data processing Basecalls were performed using bcl2fastq v2.17 for Novaseq output.
ChIP-seq reads were aligned to the UCSC mm10 genome using Bowtie 2.1 after trimming 25 nucleotides from the 3' end and in the sensitive-local mode.
Samtools v1.1 was used to convert sam files to sorted bam files
Deeptools v3.5 bamCompare function was used to convert individual bam files from each ChIP experiment to input-normalized bigwig files using the rpkm method
kentUtils v302.1 bigWigMerge was used to merge the input-normalized bigwig files into one bedgraph, sort the bedgraph file, and then convert the bedgraph to an input-normalized bigwig file using bedGraphToBigWig
Homer was used to generate tag directories from bam files of each ChIP-experiment and peaks were called using the region parameter with 125bp window size and maximum peak size of about 250bp. Peaks were filtered based on 4-fold enrichment above input with a q-value threshold of 0.01. Peaks were converted to bed files using the Homer script
Assembly: UCSC mouse mm10
Supplementary files format and content: bigwig for ChIP and ATAC, bam for ChIP input, bed for EKLF peaks determined by Homer
Submission date Aug 08, 2022
Last update date Jan 20, 2023
Contact name Kaustav Mukherjee
Phone 2122414143
Organization name Icahn School of Medicine at Mount Sinai
Department Cell, Developmental and Regenerative Biology
Lab Dr. James Bieker
Street address 1 Gustave Levy Place
City New York
State/province NY
ZIP/Postal code 10029
Country USA
Platform ID GPL24247
Series (1)
GSE210779 EKLF/Klf1 regulates erythroid transcription by its pioneering activity and selective control of RNA Pol II pause-release
BioSample SAMN30201208
SRA SRX16986538

Supplementary file Size Download File type/resource
GSM6437125_EKLF_w2-in.bam 5.5 Gb (ftp)(http) BAM
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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