|
| Status |
Public on Apr 04, 2024 |
| Title |
WGBS_E95_exMidgut_EPCAM_mCherry |
| Sample type |
SRA |
| |
|
| Source name |
Midgut endoderm
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Midgut endoderm
developmental stage: E9.5
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, and the midgut from the posterior part from lineage-traced embryos were pooled and dissociated using 0,25% Trypsin-EDTA, at 37 ºC for 10 minutes. The cells were washed with 10% FBS/DMEM, stained with EPCAM antibody in 2% FBS/HBSS with 0,5 mM EDTA (FACS buffer) at 4 ºC for 10 minutes, and then EPCAM+ cells were enriched by magnetic separation (MACS). Finally, the cells were stained with DAPI at 4 ºC for 8 minutes in FACS buffer. Cells were resuspended in FACS buffer, the indicated populations were sorted into Lysis buffer (10 mM Tris-HCl (pH=8.0), 10 mM NaCl, 10 mM EDTA, 0.5% SDS, 300 ug/mL Proteinase K) using the BD FACSAria Fusion. Genomic DNA was isolated by phenol–chloroform extraction.
Genomic DNA was sheared in micro TUBE AFA Fiber Pre-Slit Snap-Cap tubes (Covaris 520045) followed by phenol–chloroform extraction. The purified DNA was bisulfite-converted using the EZ DNA Methylation-Gold Kit (Zymo D5005), and WGBS libraries were processed using the Accel-NGS Methyl-seq DNA library kit (Swift Biosciences 30096) following the manufacturer’s recommendations. Libraries were cleaned using Agencourt AMPure XP beads. Quality and concentration of the obtained libraries were measured using Agilent High Sensitivity D5000 ScreenTape on an Agilent 4150 TapeStation. Libraries were then sequenced using 150 base pair paired-end sequencing on a NovaSeq 6000 platform.
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| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| 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 clipped from both reads), followed by trimming of 10 and 5 nucleotides from the 5’ and 3’ end of the first read and 15 and 5 nucleotides from the 5’ and 3’ end of the second read.
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 16 -q 20 -w 100 -S 1 -u -R).
Duplicates were removed using the ‘MarkDuplicates’ command from GATK (version 4.3.0.0; --VALIDATION_STRINGENCY=LENIENT --REMOVE_DUPLICATES=true).
Methylation rates were called using mcall from the MOABS package (version 1.3.2; default parameters).
Only CpGs covered by at least 10 and at most 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 10 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) |
hetzel@molgen.mpg.de
|
| 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 |
| GSE250181 |
Extraembryonic gut endoderm cells undergo programmed cell death during development (WGBS) |
|
| Relations |
| BioSample |
SAMN38839202 |
| SRA |
SRX22886230 |
