|
| Status |
Public on Nov 25, 2013 |
| Title |
Proliferating BS-seq Rep3 |
| Sample type |
SRA |
| |
|
| Source name |
proliferating cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: IMR-90
|
| Treatment protocol |
For Proliferating and Senescent cells no treatment was performed outside the library construction. To achieve senescence bypass in near senescent IMR90 a SIN human immunodeficiency virus I (lenti) vector expressing the early region (encoding Large and small T-antigen) of Simian virus 40 (SV40) from the CMV-promoter and the gene encoding aminoglycoside phosphotransferase (Neomycin resistance), driven by the SV40 early promoter was generated. After infection IMR90 were cultured in the presence of 500µg/ml G418-sulphate (Invitrogen) for one week, to select for SV40 T-antigen expressing cells.. (encoding Large and small T-antigen) of Simian virus 40 (SV40) from the CMV-promoter and the gene encoding aminoglycoside phosphotransferase (Neomycin resistance), driven by the SV40 early promoter was generated.
|
| Growth protocol |
IMR-90 cells (Coriell) were grown in Dulbecco’s Modified Eagle Medium, high glucose (Life Technologies) supplemented with 20% Fetal Bovine Serum (FBS), 2mM L-glutamine and 100ug/ml penicillin-streptomycin (Life Technologies) and incubated at 37˚C with 3% (v/v) O2 and 5% (v/v) CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted with phenol-chloroform method.
Libraries were prepared by BGI, Shenzhen. Briefly, DNA is fragmented and methylated adapters are ligated to DNA fragments. DNA is bisulphite treated and sequenced on Illumina GAIIx/HiSeq platforms as per manufacturers instructions.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Bisulphite sequencing of proliferating cells
|
| Data processing |
Basecalls performed in phred64 (solexa1.3-quals).
Sequenced reads were mapped to hg18 whole genome using bismark v0.5.1 (bowtie v0.12.7) with directional setting and chunkmbs set to 256. Remaining default settings.
Multiple reads where both ends of the fragment align to the same genomic positions on the same strand were reduced to a single instance. Reads with more than 3 methylated cytosines in non-CpG contexts are discarded.
Reads are summarised on a per-CpG basis.
DMRs are computed by filtered to CpGs binomial meth call <= 0.01FDR. A sliding window was used (2kb) starting at CpG and ending at most distant CpG within 2kb of the start position. Total G, Pooled G and Heterogenous G (difference between Total G and Pooled G) are calculated for each window and converted to P-values using the chi-square distribution. P-values are FDR corrected and adjacent windows merged where corrected P-values <= 0.05. Regions smaller than 5kb are discarded and heterogeneous regions are subtracted from the Pooled G regions. Resulting Pooled G regions are reported as DMRs.
Genome_build: hg18
Supplementary_files_format_and_content: Summary files represent the number of reads supporting methylated/unmethylated bases at each CpG sequenced in that sample (Format is: chr <tab> position <tab> methylated reads <tab> unmethylated reads). bigWig files represent the percentage methylation at a given CpG (filtered to exclude CpGs with low coverage on a per replicate basis – >=10 reads in at least one sample & >=3 read in the other) and generated using UCSC wigToBigWig. peak files represent differentially methylated regions at 5% FDR.
|
| |
|
| Submission date |
Jul 08, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter D. Adams |
| Organization name |
University of Glasgow, Beatson Institute for Cancer Research
|
| Street address |
Switchback Rd, Bearsden
|
| City |
Glasgow |
| ZIP/Postal code |
G61 1BD |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE48580 |
Genome-wide methylation maps for Proliferating and Senescent cells |
|
| Relations |
| BioSample |
SAMN02228059 |
| SRA |
SRX318534 |
| Named Annotation |
GSM1181647_Rep3.Proliferating.methpercent.bigWig |
