|
| Status |
Public on Sep 12, 2012 |
| Title |
NOMe-seq IMR90 |
| Sample type |
SRA |
| |
|
| Source name |
lung fibroblast
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: IMR90
cell type: lung fiboblast cells
|
| Growth protocol |
Cells were isolated and cultured as described (PMID 14645703)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Following trypsinization, 2 million cells were washed with 1xPBS and nuclei were extracted in 10mM Tris (pH 7.4), 10mM NaCl, 3mM MgCl2, 0.1mM EDTA, 0.5% NP-40 on ice for 10 minutes followed by centrifugation at 3000 rpm 4°C for 5 minutes. Nuclei were treated with M.CviPI and DNA was extracted and fragmented (~150bp) using Covaris. 5ug DNA was end-repaired (EpiCentre) and A’s were added to the 3’ ends using Klenow. Methylated adaptors (Illumina ME-100-0010) were ligated and no more than 6 cycles of PCR was performed. DNA was run on a 2% agarose gel and fragments between 200-500 were size selected and purified. Clustering and sequencing was performed on an Illumina HiSeq according to the manufacturer's recommendations.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
M.CviPI treated following bisulfite-convertion of genomic DNA
|
| Data processing |
Illumina Casava1.7 software used for basecalling.
IMR90 reads were mapped to hg18 whole genome using MAQ-0.7.1 with parameters -c; GBM reads were mapped to hg19 whole genome using BSMAP-2.01 with parameters -z 64 -p 11 -s 18 -v 10 -q 2
BisSNP-0.70 were used to do base quality recalibration and indel realignment with dbSNP135, 1000G_phase1 for known SNP and indels file, duplicated reads were marked by MarkDuplicates.jar in Picard
BisSNP-0.70 were used to call genotype and DNA methylation with dbSNP135 known SNPs file and parameters, -mmq 30 -mbq 5 -minConv 1 -stand_call_conf 20
DNA methylation information were extracted into wig file at GCH, HCG sites with at least 1 C or T reads
Genome_build: hg18(IMR90), hg19(2 GBMs)
Supplementary_files_format_and_content: wig files, DNA methylation information were extracted into wig file at GCH, HCG sites with at least 1 C or T reads
|
| |
|
| Submission date |
Sep 11, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Yaping Liu |
| E-mail(s) |
lyping1986@gmail.com
|
| Organization name |
Cincinnati Children's Hospital Medical Center
|
| Department |
Department of Pediatrics
|
| Lab |
EpiFluid Lab in Division of Human Genetics
|
| Street address |
3333 Burnet Ave
|
| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE40770 |
Genome-wide mapping of nucleosome positioning and DNA methylation within Individual DNA molecules |
|
| Relations |
| SRA |
SRX186031 |
| BioSample |
SAMN01164092 |
