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| Status |
Public on Jan 02, 2023 |
| Title |
ChIP-BS-seq_TSC1_WT_H3K27me3_Rep1 |
| 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
antibody: H3K27me3
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| Growth protocol |
TSCs (TSC line 1) were cultured in 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).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5 million cells were resuspended in 500 µl cell lysis buffer (20 mM Tris-HCl ph8, 85 mM KCl, 0.5% NP40) and incubated for 5 minutes on ice followed by 2500 g centrifugation at 4 ºC for 5 minutes. Supernatant was removed and pelleted nuclei were resuspended in 100 µl PBS, after which an additional 100 µl of 2x lysis buffer supplemented with 40 U/µl micrococcal nuclease (NEB) was added (100 mM Tris-HCL pH 8.0, 300 mM NaCl, 2% TritonX-100, 0.2% sodium deoxycholate, and 10 mM CaCl2). Nuclear lysis was carried out on ice for 20 minutes followed by a 25 minute incubation at 37 ºC, which was previously experimentally determined to yield mono-nucleosome sized fragments. The micrococcal nuclease reaction was terminated with the addition of 800 µl lysis dilution buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% TritonX-100, 50 mM EGTA, 50 mM EDTA, and 0.1% sodium deoxycholate) and 2 µg of H3K27me3 antibody was added (Thermo Scientific MA5-11198). Immunoprecipitation was carried out at 4 ºC overnight with gentle rotation followed by incubation with protein A dynabeads (Thermo Scientific) for 4 hours the next day. Bead bound immune complexes were washed at 4 ºC two times with RIPA buffer (0.1% DOC, 0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH 8.0, 1mM EDTA, 140 Mm NaCl), and then one time with each of the following: RIPA high salt (0.1% DOC, 0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH 8.0, 1mM EDTA, 360 mM NaCl), LiCl wash buffer (250mM LiCl, 0.5% NP40, 0.5% deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8.0), and TE pH 8.0. DNA was then eluted from the protein A beads by dissolving them in 100 µL ChIP elution buffer (TE, 0.1% SDS, and 300mM NaCl) with 0.2 mg/mL Proteinase K (Invitrogen) and incubating at 55 ºC overnight. The next day 300 µl TE was added to the reaction along with 400 µl phenol-chloroform-isoamyl alcohol (Invitrogen), the solution was briefly vortexed, and then centrifuged at 21,000 g for 10 minutes at room temperature in phase lock tubes (VWR). After centrifugation, the aqueous phase was collected and combined with 20 µL 5M NaCl, 1 µL 20 mg/mL glycogen (Thermo Scientific), and 880 µL 100% ethanol. This solution was stored at -20 ºC overnight and then centrifuged at 21,000 g 4 ºC for 1 hour the next day. This was followed by two washes with 70% ethanol and eluted in 20 µL 1x TE.
The resulting DNA was bisulfite converted with the EZ DNA methylation gold kit (Zymo) and used as input for the Accel-NGS® Methyl-Seq DNA Library Kit (Swift/IDT) following the manufacturer’s protocol. All libraries were sequenced using 100 bp paired-end sequencing (200 cycles kit) on a NovaSeq 6000 platform.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw reads of ESC and TSC H3K27me3 ChIP-BS-seq samples as well as their respective input samples were subjected to adapter and quality trimming with cutadapt (version 2.4; parameters: --quality-cutoff 20 --overlap 5 --minimum-length 25 --adapter AGATCGGAAGAGC -A AGATCGGAAGAGC).
Reads were aligned to the mouse genome (mm10) using BSMAP (version 2.90; parameters: -v 0.1 -s 16 -q 20 -w 100 -S 1 -u -R).
A sorted BAM file was obtained and indexed using samtools with the ‘sort’ and ‘index’ commands (version 1.10).
Duplicate reads were identified and removed using GATK (version 4.1.4.1) ‘MarkDuplicates’ and default parameters.
Replicates of treatment and input samples were merged respectively using samtools ‘merge’.
Methylation rates were called using mcall from the MOABS package (version 1.3.2; default parameters).
All analyses were restricted to autosomes and only CpGs covered by at least 10 and at most 150 reads were considered for downstream analyses.
Genome-wide coverage tracks for single and merged replicates normalized by library size were computed using deepTools bamCoverage (parameters: --normalizeUsing RPGC --extendReads).
Coverage tracks were subtracted by the respective input using deeptools ‘bigwigCompare’.
Assembly: mm10
Supplementary files format and content: H3K27me3: Bigwig coverage files (RPGC normalization, input subtracted).
Supplementary files format and content: DNA methylation: Bigwig file containing methylation rates for CpGs covered by at least 10 and at most 150 reads: <chr> <start> <end> <methylation rate>
Library strategy: ChIP-BS-seq
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| Submission date |
Nov 02, 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 |
| GSE217138 |
Dynamic antagonism between key repressive pathways maintains the placental epigenome (ChIP-BS-seq) |
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| Relations |
| BioSample |
SAMN31577004 |
| SRA |
SRX18128350 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6705569_ChIP-BS-seq_TSC1_WT_H3K27me3_Rep1_DNAme_mm10.bw |
13.6 Mb |
(ftp)(http) |
BW |
| GSM6705569_ChIP-BS-seq_TSC1_WT_H3K27me3_Rep1_RPGC_mm10_input_subtracted.bw |
301.6 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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