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Status |
Public on Jan 16, 2024 |
Title |
mESC, synHPRT1R integrated on chr3, clone 1, H3K27me3 ChIP, rep 2 |
Sample type |
SRA |
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Source name |
BL6xCAST with synHPRT1R integrated on chr3
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Organism |
Mus musculus |
Characteristics |
cell type: Embryonic stem cells genotype: synHPRT1R
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Growth protocol |
Cells were cultured in 80/20 medium, which consists of 80% 2i medium (1:1 mixture of Advanced DMEM/F12 (ThermoFisher 12634010) and Neurobasal-A (ThermoFisher 10888022) supplemented with 1% N2 Supplement (ThermoFisher 17502048), 2% B27 Supplement (ThermoFisher 17504044), 1% GlutaMAX™ (ThermoFisher 35050061), 1% Pen-Strep (ThermoFisher 15140122), 0.1 mM 2-mercaptoethanol (Sigma M3148), 1,250 U/mL LIF (ESGRO ESG1107l), 3 μM CHIR99021 (R&D Systems 4423), and 1 μM PD0325901 (Sigma PZ0162)), and 20% mESC medium (KnockOut™ DMEM (ThermoFisher 10829018) supplemented with 15% FBS (BenchMark 100106), 0.1 mM 2-mercaptoethanol, 1% GlutaMAX™, 1% MEM non-essential amino acids (ThermoFisher 11140050), 1% nucleosides (EMD Millipore ES-008-D), 1% Pen-Strep, and 1,250 U/mL LIF). mESCs were maintained on plates coated with 0.1% gelatin (EMD Millipore ES-006-B) at 37oC in a humidified incubator with 5% CO2.
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Extracted molecule |
genomic DNA |
Extraction protocol |
Cells were grown to medium confluency in 6-well plates. Cells were harvested by washing once with PBS, disso-ciating into single-cell suspending with TrypLE Express (ThermoFisher 12604013) and then neutralizing with mESC media. Crosslinking was performed by adding formaldehyde to a final concentration of 0.1% (v/v), and cells were incubated at room temperature for 5 minutes with occasional mixing by in-version. Crosslinking was stopped by quenching with 125 mM glycine, incubating at room temperature for 5 minutes with occasional mixing by inversion. DMSO was added to a final concentration of 10% (v/v) and cells were frozen in aliquots of ~106 cells. ~10^6 crosslinked and frozen mESCs were thawed and processed for CUT&RUN using the CUTANA™ ChIC/CUT&RUN kit (EpiCy-pher 14-1048) according to the manufacturer’s protocol. Antibodies were all used at 0.5 μg: rabbit IgG negative control (EpiCypher 13-0042), H3K4me3 (EpiCypher 13-0041), H3K27ac (EpiCypher 13-0045), H3K27me3 (Active Motif 39055, RRID: AB_2561020), Pol II (Santa Cruz Biotechnology sc-56767). Sequencing libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs E7645L) according to the manufacturer's protocol.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina NextSeq 500 |
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Data processing |
Demultiplexing and fastq generation was performed using Illumina bcl2fastq v2.20 bowtie2 (v2.2.9) was used to align reads to custom references in which the synthetic HPRT1 and HPRT1R sequences were present on separate chromosomes or inserted at their specific integration sites in the GRCm38/mm10 genome (produced using the reform tool, https://gencore.bio.nyu.edu/reform/). Samtools (v1.9) was used to sort and index bam files. Coverage tracks were produced in bigWig format using bamCoerage (deepTools v3.5.0) with options: --binSize 10 --smoothLength 100 --normalizeUsing RPGC --effectiveGenomeSize 2652783500 Assembly: GRCm38/mm10 + HPRT1/HPRT1R, GRCm38/mm10 reformed with HPRT1/HPRT1R custom genomes Supplementary files format and content: bigWig - coverage depth tracks for visualiztion
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Submission date |
Jan 03, 2024 |
Last update date |
Jan 16, 2024 |
Contact name |
Brendan R. Camellato |
E-mail(s) |
brendan.camellato@nyulangone.org
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Organization name |
NYU Langone Health
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Department |
Institute for Systems Genetics
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Lab |
Boeke Lab
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Street address |
435 E 30th St
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City |
New York |
State/province |
New York |
ZIP/Postal code |
10016 |
Country |
USA |
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Platform ID |
GPL19057 |
Series (2) |
GSE252478 |
Synthetic reversed sequences reveal default genomic states [mESC_ChIP-Seq] |
GSE252482 |
Synthetic reversed sequences reveal default genomic states |
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Relations |
BioSample |
SAMN39244453 |
SRA |
SRX23085787 |
Supplementary file |
Size |
Download |
File type/resource |
GSM8001879_mESC_HPRT1R_hHPRT1R_hHPRT1R_full_mBRC348_Sox2_2-H3K27me3-BS22258A.HPRT1RpLM1110.sort.bam.bw |
406.1 Mb |
(ftp)(http) |
BW |
GSM8001879_mESC_HPRT1R_hHPRT1R_hHPRT1R_full_mBRC348_Sox2_2-H3K27me3-BS22258A.reform.sort.bam.bw |
404.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
Raw data are available in SRA |
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