GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM4593077 Query DataSets for GSM4593077
Status Public on Oct 11, 2021
Title WT_ESC_input_TF_group3
Sample type SRA
Source name Embryonic stem cells
Organism Mus musculus
Characteristics cell type: J1 cells
genotype: WT
age: 0 days
Growth protocol Embryonic stem cells (ESCs) were cultured on 1% gelatin with a feeder layer of MEFs in DMEM supplemented with 15% Hyclone FBS (GE Healthcare), GlutaMax (Invitrogen), penicillin/streptomycin, non-essential amino acids, 1000U/mL LIF, and 2-betamercatoethanol. To form embryoid bodies (EBs), ESCs were de-MEFed and resuspended in differentiation media (IMDM supplemented with 15% HyClone FBS , GlutaMax, and penicillin/streptomycin) at a concentration of 8,400 cells/mL. Drops were set at 50uL per drop on the bottom of a non-adherent tissue culture plate, and plates were storeed upside down. After 4 days, EBs were pooled together and grown in suspension in non-adherent plates, changing media every day. EBs were collected for analysis on days 6 and 10 of differentiation.
Extracted molecule genomic DNA
Extraction protocol Ten to twenty million harvested cells were crosslinked in 1% formaldehyde-PBS for 10 minutes at room temperature. Crosslinking was quenched with glycine, and cells were pelleted by centrifugation at 4°C and washed once in cold PBS. Cells were resuspended in cold lysis buffer (50mM Tris-HCl pH 8.1, 10mM EDTA pH 8.0, 1% SDS, PIC, 1mM DTT) and incubated for 10 minutes on ice prior to sonication using a QSonica Q800R3 Sonicator. Insoluble chromatin was pelleted by centrifugation, and supernatant was diluted 1:10 with IP Dilution Buffer (20mM Tris-HCl pH 8.1, 150mM NaCl, 2mM EDTA pH 8.0, 0.1% Triton X-100, PIC, 1mM DTT). Sonicated Drosophila chromatin was added at a known ratio to experimental chromatin to serve as a spike-in as previously described (Egan et al., 2016). Triton X-100 was added to a final concentration of 1%, input was collected and diluted to 200mL, and chromatin was added to Protein A Dyanbeads (Invitrogen) that had been previously incubated with experimental and spike-in antibodies. Samples were rotated at 4°C overnight. Beads were washed with IP Dilution Buffer with 1% Triton X-100, followed by Wash Buffer A (50mM HEPES pH 7.9, 500mM NaCl, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, PIC, DTT), followed by Wash Buffer B (20mM Tris-HCl pH 8.0, 250mM LiCl, 1mM EDTA pH 8.0, 0.1% Na-deoxycholate, 1% Igepal, PIC, DTT), followed by a quick TE wash. Beads were incubated at 37°C in 200mL Extraction Buffer (1% SDS, 0.1M NaHCO3) for 15 minutes with shaking. Supernatant was collected, and 12mL 2M TrisHCl, 2mL RNase A (500mg/mL, Roche), and 12mL 5M NaCl were added to ChIP samples and inputs. Crosslinks were reversed overnight at 65°C, followed by 1 hour incubation at 55°C with Proteinase K. DNA was isolated by phenol:chloroform extraction and ethanol precipitation.
Libraries were prepared as described in Bowman et al., 2013 (BMC Genomics). In brief, this protocol includes end repair, A-tailing, adapter ligation, and real-time PCR amplification, with SPRI cleanup of samples between each step.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
Data processing Adapter sequences were removed from reads usinng Cutadapt v.1.14 with "--minimumLength 30"
Reads were aligned to the mm10 and dm6 genomes using Bowtie 2 v. with one mismatch permitted per seed, a maximum insert of 2kb, and the "--no-discordant" option specified
PCR duplicates were removed using Samtools v1.9
To normalize ChIP-Seq data, mm10 reads were randomly downsampled using Samtools. The downsampling factor reflected the total number of dm6 reads in the sample, and was corrected with the ratio of dm6:mm10 reads to account for variation in spike-in mixing.
Genome_build: mm10
Supplementary_files_format_and_content: Genome browser tracks were generated using Homer v4.10.4
Supplementary_files_format_and_content: Peak files were generated using Homer v4.10.4. Peaks were filtered to those within 10kb of their associated TSS, and to a minimum tag density that was determined by examination of IGV browser tracks.
Submission date Jun 05, 2020
Last update date Oct 12, 2021
Contact name Robert Kingston
Organization name Massachusetts General Hospital
Department Molecular Biology
Lab Kingston Lab
Street address 185 Cambridge Street, 7th Floor
City Boston
State/province MA
ZIP/Postal code 02114
Country USA
Platform ID GPL13112
Series (2)
GSE151899 A Polycomb domain found in committed cells impairs differentiation when introduced into PRC1 in pluripotent cells (ChIP-seq, CUT&RUN datasets)
GSE151901 A Polycomb domain found in committed cells impairs differentiation when introduced into PRC1 in pluripotent cells
BioSample SAMN15147092
SRA SRX8480702

Supplementary file Size Download File type/resource
GSM4593077_WT_ESC_input_TF_group3.bigWig 163.0 Mb (ftp)(http) BIGWIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap