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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 01, 2019 |
| Title |
BRAF1_EV [BiSulfite-seq] |
| Sample type |
SRA |
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| Source name |
Emty vector control organoids cultured 5 months and further grown in full medium for 3 weeks
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| Organism |
Mus musculus |
| Characteristics |
culture time: 5 months
organoids: control
medium: full
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| Growth protocol |
Male mice from each genotype around the age of 40 days were euthanized by Carbon Dioxide. The first 2.5cm of proximal colon is collected from the mice. The colon crypts were isolated and the culture was established as described previously. Organoids were plated within Matrigel(Corning #356231). Culture media was made of 50% of Wnt3a conditioned media, 20% of R-Spondin conditioned media, 20% of Advanced DMEM/F12(ThermoFisher #12634010) with B27® (ThermoFisher #17504044), 10% of Noggin conditioned media and EGF. The conditioned media for Wnt3a, R-Spondin and Noggin were made as described previously.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA/RNA was extract using Allprep kit mini or micro (Qiagen)
RRBS was performed as previously reported (Petkovich et al., 2017). RRBS library was constructed following exactly the same protocol. Sequencing libraries were sequenced on the Illumina HiSeq 2500 platform using 50bp single-end sequencing with v4 reagents (Bauer Core Facility, Harvard University). Since RRBS libraries generate low complexity libraries, the samples were spiked with ∼10% phiX.
Ligation reactions were set up by adding 4.8 µl of T4 Ligation buffer, 1.6 µl of T4 DNA Ligase (New England Biolabs), and 3.2 of µl TruSeq Nano DNA LT adapter from Illumina that was diluted 1:20, and 6.4 µl of nuclease-free water to the DNA samples. We used 4–6 different indexed adaptors per library.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Control organoids grown in full medium
BRAF1_Base_Vs_BRAF1_EV_Hyper.txt, BRAF1_Base_Vs_BRAF1_EV_Hypo.txt
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| Data processing |
The quality of the read sequence was examined using the quality control package “FastQC v0.11.7”. The sequence reads were then subjected to adaptor removal and quality trimming using Trim Galore v0.4.4_dev, which comes with additional functionality for the MspI-digested RRBS libraries. Alignment of the sequenced reads was performed using Bismark v0.14.3, and the identified methylation sites were analyzed using the Bismark processing report (Krueger and Andrews, 2011). Guidance from the “M-Bias Plot” was used to further trim the biased methylation sites and the reads were realigned using Bismark (Hansen et al., 2012). For the downstream analysis, “Methylkit” package version 1.7.4 in R platform was used (Akalin et al., 2012). A minimum read coverage of 4 and a minimum quality of 20 was used to call a methylation status of the base. The mouse genome, gene annotations and locations of CpG islands were obtained from the UCSC Genome Browser with the assembly GRCm38/mm10. Differentially methylated regions (DMRs) were identified by using tiling windows of 200 base pairs, minimum average methylation difference of 20 percent and a q-value threshold of 0.01. DMRs were associated with the annotated genomic intervals using the package “Genomation v1.13.0” (Akalin et al., 2015). Statistical significance of the overlap between two groups of methylated genes of various samples were calculated using the website http://nemates.org/MA/progs/overlap_stats.html.
Genome_build: mm10
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| Submission date |
Oct 02, 2018 |
| Last update date |
Jan 01, 2019 |
| Contact name |
hari easwaran |
| Organization name |
Johns Hopkins University
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| Department |
Oncology
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| Lab |
Steve Baylin
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| Street address |
1650 Orleans Street, CRB1, Room 530
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| City |
BALTIMORE |
| State/province |
MD |
| ZIP/Postal code |
21209 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE114801 |
Trancriptomic and epigenetic analyses of mouse organoids expressing BrafV600E mutation |
| GSE120759 |
Trancriptomic and epigenetic analyses of mouse organoids expressing BrafV600E mutation [BiSulfite-seq] |
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| Relations |
| BioSample |
SAMN10166827 |
| SRA |
SRX4789714 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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