|
| Status |
Public on Jan 01, 2016 |
| Title |
v6.5 knock-down oxBS-seq replicate 3 |
| Sample type |
SRA |
| |
|
| Source name |
parental V6.5 mES cells_oxBS-seq
|
| Organism |
Mus musculus |
| Characteristics |
cell line: V6.5
cell type: embryonic stem cells
genotype/variation: wild-type
|
| Treatment protocol |
Cells stably depleted of Tet2 was obtained by electroporating V6.5 mESC with pSUPER-puro-Tet1shRNA or pSUPER-puro-Tet2shRNA (320V, 250F). Cells were selected by 1.5 mg/ml puromycin for 7-10 days on puromycin-resistant mitomycine C-inactivated SNL76/7-4 feeder cells. Individual clones were picked and propagated in the absence of puromycin if the passage number was less than 10, or maintained in 1 mg/ml puromycin if the passage number was longer than 10. Tet2 mRNA expression in individual clones was evaluated by qPCR.
|
| Growth protocol |
Mouse embryonic stem cells were cultured in Knockout D-MEM with 15% ES-qualified FBS (Gemini Bio-products), 2 mM L-glutamine with 0.1 mM 2-mercaptoethanol, 0.1 mM nonessential amino acids, 50 units/ml penicillin/streptomycin and 1000 U/ml ESGRO® (LIF; Chemicon).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA were extracted using Qiagen DNeasy Blood and tissue kit by following manufacture’s instruction.
Purified genomic DNA were equally split into two. One part were treated with KRuO4 following the protocol from PMID: 24008380. Then both genomic DNA were parallelly treated with sodium bisulfite using MethylCode bisulfite conversion kit (Life Technologies). Targeted regions were amplified using PyroMark PCR kit and cleaned up with MinElute reaction cleanup kit (Qiagen). Amplified fragments were multiplexed using Illumina TrueSeq DNA library preparation kit. Pooled libraries were sequenced using MiSeq sequencing instrument (Illumina).
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
First the sequencing adapters were removed from the reads when encountered.
Bismark v0.7.12 (Krueger and Andrews 2011) was used to align the BS and oxBS reads against the mm9 reference genome and lambda phage DNA simultaneously. The alignment was done using the paired-end Bowtie 2 (Langmead and Salzberg 2012) backend with the following parameters ”-I 0 -X 2000 -N 0”.
The “bismark_methylation_extractor” script distributed with the Bismark aligner was used to extract the number of unconverted and converted read-outs for each cytosine with the following parameters “--paired-end --CX --cutoff 10 --no_overlap --bedGraph --counts”. The cytosines having at least 10 read-outs across all six samples were taken into account.
Genome_build: mm9
Supplementary_files_format_and_content: The processed data files have been generated by the bismark_methylation_extractor script, which is part of the Bismark software package.
The column headers are:<chromosome> <start position> <end position> <methylation percentage> <count methylated> <count unmethylated>
as described in http://www.bioinformatics.babraham.ac.uk/projects/bismark/Bismark_User_Guide.pdf.
|
| |
|
| Submission date |
May 05, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Tarmo Äijö |
| Organization name |
Flatiron Institute
|
| Department |
Center for Computational Biology
|
| Street address |
162 5th Avenue
|
| City |
New York |
| ZIP/Postal code |
10010 |
| Country |
USA |
| |
|
| Platform ID |
GPL16417 |
| Series (2) |
| GSE68574 |
A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways [mESCs] |
| GSE68576 |
A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways |
|
| Relations |
| BioSample |
SAMN03603950 |
| SRA |
SRX1018482 |
