|
| Status |
Public on Mar 19, 2024 |
| Title |
buccal DNA 356 |
| Sample type |
genomic |
| |
|
| Source name |
buccal swab
|
| Organism |
Homo sapiens |
| Characteristics |
Sex: M
age days: NA
dataset: m60
smoking: 1
|
| Treatment protocol |
Patients were instructed not to eat or drink at least 30 minutes before buccal sample collection. Buccal DNA was collected using 4 Sterile HydraFlock® Large Tip Flocked Swabs (Puritan 253406H). Each swab was applied to the inside of the child’s cheek and rolled up and down for 30-60 seconds, making certain to roll the swab over the entire inner cheek and upper gum. Swabs were placed together in a 15 ml conical tube containing 1600ul of 1X Saliva Lysis Buffer (SLB) before capping tube and vortexing for 10 seconds. The swabs were frozen upright in SLB at -20°C within 1 week from sampling and stored until DNA extraction. 2X Saliva Lysis Buffer: 0.3 M TRIS-HCl; 0.67 M urea; 0.67 M NaOAc; 0.6% sodium dodecyl sulfate; 3.3 mM EDTA; 30% ethanol)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Buccal DNA was extracted using the Maxwell® 16 Instrument (Promega AS3000) and Maxwell® 16 Blood DNA Extraction Kit (Promega AS1010). Proteinase K (Promega 20mg/ml) was added to 15ml conicals containing 4 swabs and ~1.6ml SLB. Tubes were vortexed to collect SLB at bottom. Tubes were incubated in s 56°C water bath for 1 hr and then vortexed. Lysate (800ul) was added to well #7 of the Maxwell® 16 Blood DNA cartridge. The remaining lysate was stored for a second DNA extraction. Alcohol Wash Buffer (2 parts Milli-Q water, 1 part 100% EtOH, 1 part 100% isopropanol) was added to wells #4, #5 and #6. Cartridges were processed using the Buffy Coat Protocol on the Maxwell® 16 Instrument and eluted in 400ul elution buffer. Purity was measured by spectrophotometer and concentration measured with the Quant-iT PicoGreen dsDNA Assay (Invitrogen® P7589).
|
| Label |
Cy3 and Cy5
|
| Label protocol |
none provided
|
| |
|
| Hybridization protocol |
none provided
|
| Scan protocol |
Buccal DNAm was assessed on the MethylationEPIC BeadChips, measuring over 850,000 CpGs at a nucleotide resolution, at the Fred Hutchinson Cancer Research Center Genomics Resource. In brief, 500ng of DNA was bisulfite converted using the EZ DNA Methylation Kit (Zymo Research) following the Illumina-specified instructions. Converted DNAs were applied to Illumina Infinium Methylation EPIC 8-Sample Beadchips following the Infinium HD Methylation 15019521v01 protocol. Processed BeadChips were scanned using the Illumina iScan+ with ICS v3.3.28 and intensity data was extracted with Illumina GenomeStudio software (GenomeStudio v2011.1 with Methylation Analysis Module v1.9.0).
|
| Data processing |
Data normalization and QC were performed using ChAMP: non-CpG probes, probes with a beadcount <3 in at least 5% of samples, probes annotated to SNPs, probes with a detection p-value > 0.01 in one or more samples, cross-hybridizing probes, and probes on X/Y chromosomes were removed and remaining probes (n=746,421) were normalized using via functional normalization.
functional normalized beta
Unmethylated and methylated signal intensities
|
| |
|
| Submission date |
Dec 27, 2023 |
| Last update date |
Mar 19, 2024 |
| Contact name |
Lyndsey Elizabeth Shorey-Kendrick |
| E-mail(s) |
shorey@ohsu.edu
|
| Phone |
5033465517
|
| Organization name |
Oregon Health and Science University
|
| Street address |
505 NW 185th Ave
|
| City |
Beaverton |
| State/province |
Oregon |
| ZIP/Postal code |
97006 |
| Country |
USA |
| |
|
| Platform ID |
GPL21145 |
| Series (1) |
| GSE252169 |
Epigenome-wide profiling of buccal DNAm in samples obtained from children of pregnant smokers enrolled in the VCSIP RCT: NCT03203603 |
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