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| Status |
Public on Jan 02, 2023 |
| Title |
MINUTE_TSC1_WT_H2AK119ub1_Pool1_Rep3 |
| Sample type |
SRA |
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| Source name |
Trophoblast stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Trophoblast stem cells
condition: WT
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| Treatment protocol |
On the day of collection, TSCs were rinsed once with DPBS (Thermo, #14190144) and detached using Trypsin-EDTA (0.05%)(Thermo, #25300054). The cells were spun down for 4 min at 1000 rpm, counted and flash frozen on dry ice.The cell pellets were stored at -80°C.
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| Growth protocol |
Trophoblast stem cells (TSCs) cells were grown on MEF-coated cell culture dishes containing TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149). Splitting was carried out every five to seven days by rinsing the cells once with DPBS (Thermo, #14190144) before detaching the cells using Trypsin-EDTA (0.05%)(Thermo, #25300054). TSCs were passaged in clumps. Before sample collection, TSCs were passaged at least one passage without MEFs to dilute out feeder cells. During this time, the cells were cultured in MEF-conditioned TSC medium (70 % MEF-conditioned medium, 30 % TSC medium, +FGF4 (37.5 ng/ml), Heparin (1.5 µg/ml)).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Native cell pellets were lysed and digested with MNase to enrich for mononucleosome population. The digestion was quenched by EGTA-containing end-repair and ligation buffer, in which each sample was ligated to adaptor molecules carrying unique barcode. Ligation was quenched by EDTA-containing lysis dilution buffer, before combining all samples in one tube. After centrifugation, pool supernatant was recovered and aliquoted for individual ChIP. 2 x 10^6 cell-equivalent of pool supernatant was used for ChIP against each histone modification, with the anti-H3K4me3 (Millipore #04-745), anti-H3K27me3 (Cell Signaling #9733) and anti-H2AK119ub (Cell Signaling #8240) antibodies precoupled to Protein A magnetic beads.
After thorough washes, ChIP DNA was recovered with Proteinase K treatment and purified for linear amplification by in vitro transcription. The RNA product was then ligated to a pre-adenylated RNA 3’ adaptor (RA3), which served as a primer binding site for reverse transcription. The resulting cDNA was purified and used as a template for library PCR with barcoded primers compatible with Illumina sequencing platform. Typically, 1-2 x 10^5 cell equivalent of pool supernatant was used as Input, which is subjected to the same experimental workflow for library construction as the ChIP DNA. All nucleic-acid purification were carried out with AMPure SPRI size selection method (Beckman Coulter). Library size distribution was assessed by Agilent BioAnalyzer and were quantified by Qubit DNA high sensitivity assay before dilution for sequencing on a NovaSeq 6000 platform.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Quantitative ChIP of TSCs
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| Data processing |
Read pairs matching parts of the adaptor sequence (SBS3 or T7 promoter) in either read1 or read2 were removed using cutadapt v3.2.
Reads were demultiplexed using cutadapt v3.2 allowing only one mismatch per barcode and written into sample-specific FASTQ files used for subsequent mapping.
Sample-specific paired FASTQ files were mapped to the reference mm10 using bowtie2 v2.3.5.1 with --fast and --reorder parameter. Alignments were processed into sorted BAM files and replicates were pooled using samtools v1.10.
Duplicate reads are marked using UMI-sensitive deduplication tool je-suite (v2.0.RC) (https://github.com/gbcs-embl/Je/). Read pairs are marked as duplicates if their read1 (first-in-pair) sequences have the same UMI (allowing for 1 mismatch) and map to the same location in the genome. Blacklisted regions as downloaded from ENCODE were then removed from BAM files using bedtools v2.30.
Input coverage tracks with 1 bp resolution in bigwig format were generated from BAM files using deepTools v3.5.0 bamCoverage and scaled to a reads-per-genome-coverage of one (1xRPGC, also referred to as ‘1x normalization’) using the mm10 effective genome size. ChIP coverage tracks were generated from BAM files using deepTools (v3.5.0) bamCoverage. Quantitative scaling of the ChIP-Seq tracks amongst conditions within each pool was based on their Input-Normalized Mapped Read Count (INRC). INRC was calculated by dividing the number of unique reference-mapped reads by the respective number of Input reads: #mapped[ChIP] / #mapped[Input]. This essentially corrects for an uneven representation of barcodes in the Input. It has been previously shown that INRCs are proportional to the amount of epitope present in each condition (8). Reference condition (TSC WT) was scaled to 1x coverage (also termed Reads per Genome Coverage, RPGC). All other conditions within the same pool were scaled relative to the reference using the ratio of INRCs multiplied by the scaling factor determined for 1x normalization of the reference: ( #mapped[ChIP] / #mapped[Input] ) / ( #mapped[ChIP_Reference] / #mapped[Input_Reference] ) * scaling factor.
Genome_build: mm10
Supplementary_files_format_and_content: Bigwig files are scaled according to MINUTE quantification with respect to the reference of each pool (TSC1 WT, 1xRPGC)
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| Submission date |
Mar 07, 2022 |
| Last update date |
Jan 02, 2023 |
| Contact name |
Sara Hetzel |
| E-mail(s) |
hetzel@molgen.mpg.de
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| Organization name |
Max Planck Institute for Molecular Genetics
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| Department |
Genome Regulation
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| Lab |
Meissner Lab
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| Street address |
Ihnestraße 63
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| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE166362 |
Dynamic antagonism between key repressive pathways maintains the placental epigenome |
| GSE198075 |
Dynamic antagonism between key repressive pathways maintains the placental epigenome (MINUTE-ChIP) |
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| Relations |
| BioSample |
SAMN26511533 |
| SRA |
SRX14403042 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5938105_MINUTE_TSC1_WT_H2AK119ub1_Pool1_Rep3_RPGC_mm10_scaled.bw |
103.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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