 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 03, 2019 |
| Title |
WGBS_PBL_E018 |
| Sample type |
SRA |
| |
|
| Source name |
PBL
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Female
tissue origin: Blood
|
| Treatment protocol |
NA
|
| Growth protocol |
NA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
SA and VA samples - In brief, freshly isolated VAT and SAT samples were weighed and minced with surgical scissors. For each gram of minced tissue, 1.25 mg/mL of Collagenase I (Sigma Aldrich, CAT#C6885-1G) was added to 2 mL of HEPES buffer containing 7.9 g/L NaCl, 323 mg/L CaCl2.2H2O, 308 mg/L MgSO4.7H2O, 154.3 mg/L Na2HPO4 (anhydrous), 5 mL/L of 1M Hepes (Sigma Aldrich), 450 mg/L D-glucose, pH 7.2 – 7.4, 20 g/L BSA. The tissue was incubated for 30-45 minutes at 37ºC before being diluted 1:9 with additional HEPES buffer. The mixture was passed through a 250 micron mesh before being centrifuged at 300 rcf for 6 minutes at room temperature. The top lipid layer was removed and the adipocytes collected for snap-freezing in liquid nitrogen.; VATsamples - Snap frozen in liquid Nirtogen; PBL - 10 mL blood was collected into K2EDTA vacutainers. Following centrifugation at 1,300 rcf for 10 minutes at room temperature, the top plasma layer was removed and the buffy coat layer collected. Cells were washed three times, with vigorous resuspension, in 10 mL Tris-EDTA buffer (100 mM Tris-HCl, 0.1 mM EDTA), with pellets collected after centrifugation at 10,000 rcf for 10 minutes. The final pellet was resuspended in 500 µL of buffer and stored at -80ºC. DNA was extracted: SAVA - using proteinase K digestion and phenol chloroform extraction; VAT - DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Cat#69504); PBL - DNA was extracted from PBL sample types following the Gentra Puregene Blood Cell Kit (Qiagen, Cat#158445).
Whole genome bisulfite sequencing libraries were prepared following Illumina’s “Whole- Genome Bisulfite sequencing for Methylation Analysis” protocol. Briefly, 1 mg of genomic DNA was spiked with 0.5% unmethylated lambda DNA and sonicated to generate fragments of size between 150 to 300bp. Library preparation was performed for core samples using the Illumina’s Paired-end DNA Sample Prep Kit (discontinued, Illumina, CA, USA) according to the manufacturer’s protocol. The size selected libraries were then subjected to bisulfite conversion as previously described {Clark, 2006 #385}. Adaptor-ligated bisulfite treated DNA was enriched by 10 cycles of PCR amplification using the PfuTurbo Cx Hotstart DNA Polymerase (Stratagene). Qualitative and quantitative checks of the libraries were performed using Agilent’s High sensitivity DNA kit (Agilent) and KAPA Library quantification kit (KAPA Biosystems).
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
E18BUF
Blood
|
| Data processing |
Paired-end reads were adaptor and quality score trimmed using trim_galore v0.2.8. Trimmed reads were mapped to the hg19 genome build using bismark v0.8.3 {Krueger, 2011 #519} with the parameters “‐‐bowtie2 ‐X 1000“, technical replicates pooled using samtools v0.1.19 “merge” and PCR duplicates marked using Picard v1.91 “MarkDuplicates”. Methylation count data is extracted using bismark_methylation_extractor with the parameters “‐‐comprehensive ‐‐merge_non_CpG ‐‐bedgraph ‐‐counts ‐‐report ‐‐gzip ‐‐buffer_size 20G”.
Trimmed reads were mapped to the hg19 genome build using bismark v0.8.3 {Krueger, 2011 #519} with the parameters “‐‐bowtie2 ‐X 1000“, technical replicates pooled using samtools v0.1.19 “merge” and PCR duplicates marked using Picard v1.91 “MarkDuplicates”. Methylation count data is extracted using bismark_methylation_extractor with the parameters “‐‐comprehensive ‐‐merge_non_CpG ‐‐bedgraph ‐‐counts ‐‐report ‐‐gzip ‐‐buffer_size 20G”.
Methylation count data is extracted using bismark_methylation_extractor with the parameters “‐‐comprehensive ‐‐merge_non_CpG ‐‐bedgraph ‐‐counts ‐‐report ‐‐gzip ‐‐buffer_size 20G”.
Genome_build: hg19
|
| |
|
| Submission date |
Mar 06, 2018 |
| Last update date |
Jul 03, 2019 |
| Contact name |
Stephen Bradford |
| Organization name |
Garvan Institute of Medical Research
|
| Street address |
384 Victoria St
|
| City |
Darlinghurst |
| State/province |
NSW |
| ZIP/Postal code |
2010 |
| Country |
Australia |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE110821 |
Developmental origins define epigenomic differences between subcutaneous and visceral adipocytes |
| GSE111445 |
Developmental origins define epigenomic differences between subcutaneous and visceral adipocytes [Bisulfite-Seq] |
|
| Relations |
| BioSample |
SAMN08637924 |
| SRA |
SRX3766946 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
