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Sample GSM4593073 Query DataSets for GSM4593073
Status Public on Oct 11, 2021
Title WT_ESC_RING1B_ChIP_group2
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.2.3.4.3 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
E-mail(s) kingston@molbio.mgh.harvard.edu
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
Relations
BioSample SAMN15147094
SRA SRX8480698

Supplementary file Size Download File type/resource
GSM4593073_WT_ESC_RING1B_ChIP_group2.bigWig 28.1 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

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