|
| Status |
Public on Apr 04, 2024 |
| Title |
RRBS_E135_emColon_EPCAM_GFP_WT_Rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Colon endoderm
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Colon endoderm
developmental stage: E13.5
strain: mCherry+ morula (CD1/G4) aggregated with GFP+ mESCs (G4)
genotype: WT
|
| Treatment protocol |
Embryos were generated by diploid complementation assay with aggregation. In each depression of the aggregation plate, one small GFP+ mESC colony of about 8-15 cells was added together with one zona pellucida-free mCherry+ pre-compaction morula.
|
| Growth protocol |
GFP+ mESCs were cultured on mitotically inactive primary mouse embryo fibroblasts in serum/LIF condition for two days. Aggregated embryos, cultured in KSOM, that progressed to the expanded blastocyst stage were transferred to a pseudo-pregnant CD1 female. Post-implantation embryos were isolated at the indicated stage, including a 24-hour developmental delay due to the uterine transfer procedure.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Extraembryonic tissues were removed, the intestine was dissected from a single embryo, and split into the small intestine and colon parts with a micro knife. The colon was dissociated using 0,25% Trypsin-EDTA, at 37 ºC for 10 minutes. The cells were washed with 10% FBS/DMEM, stained first with EPCAM antibody then with DAPI in 2% FBS/HBSS with 0,5 mM EDTA (FACS buffer) at 4 ºC for 10 and 8 minutes, respectively. Cells were resuspended in FACS buffer, the indicated populations were sorted into Buffer RLT plus using the BD FACSAria Fusion, and the samples were stored at -80 ºC.
RRBS libraries were prepared as part of the Smart-RRBS protocol by Gu, H et al. 2021 Nature Protocols, with the adaptation to low input bulk samples instead of single-cell input by reducing the bisulfite-converted DNA amplification cycle number according to the input. The genomic DNA was digested with both MspI and HaeIII.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Raw reads were subjected to adapter and quality trimming using cutadapt (version 4.1; parameters: --quality-cutoff 20 --overlap 5 --minimum-length 25; Illumina TruSeq adapter).
The trimmed reads were aligned to the mouse genome (mm10 including GFP and mCherry transgenes) using BSMAP (version 2.90; parameters: -v 0.1 -s 12 -q 20 -w 100 -S 1 -u -R -D C-CGG).
Methylation rates were called using mcall from the MOABS package (version 1.3.2; default parameters).
Due to the overall low coverage of low-input RRBS replicates, the replicates were combined at the raw count level of methylated and unmethylated CpGs and merged methylation rates were calculated subsequently to increase the coverage.
Only CpGs covered by at least five (single RRBS replicates) or 10 (merged RRBS replicates) and at maximum 150 reads were considered for downstream analyses.
Assembly: mm10 (including mCherry and GFP transgenes)
Supplementary files format and content: Bigwig file containing methylation rates for CpGs covered by at least 5 and at most 150 reads: <chr> <start> <end> <methylation rate>
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| |
|
| Submission date |
Dec 14, 2023 |
| Last update date |
Apr 04, 2024 |
| Contact name |
Sara Hetzel |
| E-mail(s) |
[email protected]
|
| Organization name |
Max Planck Institute for Molecular Genetics
|
| Department |
Genome Regulation
|
| Lab |
Meissner Lab
|
| Street address |
Ihnestraße 63
|
| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE250084 |
Extraembryonic gut endoderm cells undergo programmed cell death during development |
| GSE250180 |
Extraembryonic gut endoderm cells undergo programmed cell death during development (RRBS) |
|
| Relations |
| BioSample |
SAMN38839599 |
| SRA |
SRX22886019 |
