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| Status |
Public on Jan 02, 2023 |
| Title |
RRBS_TSC2_WT_DNMT1i_7d |
| Sample type |
SRA |
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| Source name |
Trophoblast stem cells
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| Organism |
Mus musculus |
| Characteristics |
condition: WT
treatment: DNMT1i
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| Treatment protocol |
TSCs (TSC line 2) 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) for 7 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.
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| Growth protocol |
TSCs (TSC line 2) 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) for 7 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The DNA was extracted using the PureLink Genomic DNA Mini Kit (Thermo, #K182002) according to manufacturer’s instructions.
Concentration of genomic DNA (gDNA) was quantified using a Qubit 3.0 Fluorometer. RRBS was performed on 100 ng gDNA of each sample using the NuGen Ovation® RRBS Methyl-Seq System (Tecan, #0353) following the manufacturer’s recommendations with the following modifications (6). After the final repair step, the bisulfite conversion of DNA was conducted using the Qiagen EpiTect Fast Bisulfite Conversion kit (Qiagen, #59824) following the manufacturer’s recommendations, eluting the bisulfite converted DNA in 23 µl EB. Libraries were amplified with 12 cycles of PCR. Amplified library purification with Agencourt RNAclean XP beads (Beckman Coulter, #A63987) was performed twice (1X). The purified libraries were quality-assessed on an Agilent 4150 Tape-Station HS D1000 ScreenTape and sequenced for 100-bp single-end reads on the NovaSeq6000 (Illumina).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Bisulfite-converted DNA
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| Data processing |
Raw reads were subjected to adapter and quality trimming using cutadapt (version 2.4; parameters: --quality-cutoff 20 --overlap 5 --minimum-length 25; Illumina TruSeq adapter), followed by NuGEN diversity adapter trimming (https://github.com/nugentechnologies/NuMetRRBS).
The trimmed reads were aligned to the mouse genome (mm10) using BSMAP (version 2.90; parameters: -v 0.1 -s 12 -q 20 -w 100 -S 1 -u -R -D C-CGG).
Aligned reads were deduplicated based on unique molecular identifiers (UMIs) using NuDup (https://github.com/nugentechnologies/nudup; parameters: --start 6 --length 6). 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 maximum 150 reads were considered for downstream analyses.
Genome_build: mm10
Supplementary_files_format_and_content: Bigwig file containing methylation rates for CpGs covered by at least 10 and at most 150 reads: <chr> <start> <end> <methylation rate>
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| Submission date |
Feb 17, 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 |
| GSE196976 |
Dynamic antagonism between key repressive pathways maintains the placental epigenome (RRBS) |
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| Relations |
| BioSample |
SAMN26027485 |
| SRA |
SRX14213887 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5906174_RRBS_TSC2_WT_DNMT1i_7d_mm10.bw |
25.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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