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| Status |
Public on Jul 13, 2023 |
| Title |
org_fetal_rep1_bisulfite (small intestinal epithelium derived organoids) |
| Sample type |
SRA |
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| Source name |
small intestinal epithelium derived organoids
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| Organism |
Mus musculus |
| Characteristics |
developmental state: E16.5
strain: C57BL/6J
treatmet: untreated
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| Growth protocol |
Fetal spheroids and adult organoids derived from E16.5 fetal epithelial fragments or scraped adult crypts harvested from proximal small intestine using 2mM EDTA were embedded in Matrigel droplets and cultured in advanced DMEM/F12 with GlutaMAX and Penicillin/Streptomycin supplemented with 50 ng/ml EGF, 100 ng/ml Noggin, and 500 ng/ml, R-Spondin1, 3 µM CHIR99021 and 10 mM Nicotinamide.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA of fetal (3 independent biological replicates) and adult organoids (3 independent biological replicates) was purified using Genomic DNA Purification Kit (Thermo scientific)
Libraries were prepared with ACCEL-NGS METHYL-SEQ DNA Library Kit.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Reads were quality trimmed with Trim Galore.
Reads were mapped and methylation status was determined with Bismark, which bisulfite converts the reads in silico prior to mapping with Bowtie2 to a bisulfite converted reference genome. (Krueger and Andrews, 2011). The mapping efficiency was low due to large number of chimeric reads. To mitigate this, the mapping was performed in two steps: first all reads were mapped in paired end mode with the option --unmapped, following which the unmapped R1 were mapped in single-end mode (directional mode) and the unmapped R2 in single-end mode (--pbat mode). The paired-end and single-end alignments were merged together after methylation extraction.
To segregate the genome into PMDs and non-PMDs, we use MethylSeekR (Burger et al., 2013). We identified a consensus set of PMDs present in all 6 samples. Fetal and adult state specific PMDs were defined as regions present in all replicates in one condition and none of the replicates in the other condition. We identified differentially methylated regions by BSmooth implemented in bsseq (Hansen et al., 2012). We used the methylation calls processed with Bismark and performed the smoothing and differential methylation analysis with BSmooth. We used only CpGs which were covered by more than four reads in all six samples. The smoothing was done for regions containing at least 70 CpG or were at least 2kb wide, whichever is largest, using the whole chromosomes as the smoothing cluster. For computing t-statistics, the variability was estimated based on the fetal samples. For identifying differentially methylated regions we used a cutoff 4.6, based on t-statistic quantiles.
Integrative Genomics Viewer (IGV) viewable TGF where the score represents percentage of methylated cytosines per-base CpG.
Genome_build: mm10
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| Submission date |
Dec 14, 2020 |
| Last update date |
Jul 13, 2023 |
| Contact name |
Laura Maarit Pikkupeura |
| Organization name |
University of Copenhagen
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| Department |
BRIC
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| Lab |
Jensen lab
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| Street address |
Ole Maaloes vej 5
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| City |
Copenhagen N |
| ZIP/Postal code |
2200 |
| Country |
Denmark |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE163191 |
Transcriptional and epigenomic profiling identifies YAP signaling as a key regulator of intestinal epithelium maturation [II] |
| GSE228519 |
Transcriptional and epigenomic profiling identifies YAP signaling as a key regulator of intestinal epithelium maturation |
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| Relations |
| BioSample |
SAMN17078588 |
| SRA |
SRX9684292 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4974286_Methyl.F1.bedgraph.gz.tdf |
119.6 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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