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Sample GSM4593080 Query DataSets for GSM4593080
Status Public on Oct 11, 2021
Title WT_NPC6_input_TF_group3
Sample type SRA
Source name Neural progenitor cells
Organism Mus musculus
Characteristics cell type: J1 cells
genotype: WT
age: 6 days
Growth protocol Neural progenitor cells were formed as described in Bibel et al., 2007 (Nature Protocols). In brief, embryonic stem cells were de-MEFed and grown in suspension for 4 days without LIF to form cellular aggregates. After 4 days, retinoic acid was added to a final concentration of 5uM for neural differentiation. NPCs were collected for analysis on days 6 and 8 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 SAMN15147085
SRA SRX8480705

Supplementary file Size Download File type/resource
GSM4593080_WT_NPC6_input_TF_group3.bigWig 263.7 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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