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| Status |
Public on Jan 02, 2023 |
| Title |
HEI193-12.5-1 |
| Sample type |
genomic |
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| Source name |
schwannoma
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEI193
treatment: cell_culture_12.5Gy
experimental design: classification
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| Growth protocol |
Patient resection specimen
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA were isolated from tumor samples using the All-Prep Universal Kit (QIAGEN, Valencia, CA)
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| Label |
Cy5 and Cy3
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| Label protocol |
Genomic DNA (1000 ng) is bisulfite converted using the Zymo EZ DNA methylation kit (Zymo Research, Irvine, CA) according to the manufacturer’s recommendations. The amount of bisulfite-converted DNA as well as the completeness of bisuflite conversion for each sample are assessed using a panel of MethyLight-based real-time PCR quality control assays as described previously (PMID: 18987824). Bisulfite-converted DNA is then used as a substrate for the Illumina EPIC BeadArrays, as recommended by the manufacturer and first described in Moran, 2016 (PMID: 26673039). Specifically, each sample is whole genome amplified (WGA) and then enzymatically fragmented.
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| Hybridization protocol |
Samples are then hybridized overnight to an 8-sample BeadArray, in which the WGA-DNA molecules anneal to locus-specific DNA oligomers linked to individual bead types. The oligomer probe designs follow the Infinium I and II chemistries, in which base extension follows hybridization to a locus-specific oligomer. With respect to the Infinium I probes, there are two different bead types for each locus, one with an oligomer that anneals specifically to the methylated version of the locus, while the other oligomer anneals to the unmethylated version of the locus. The probes terminate complementary to the interrogated CpG site for methylated loci, or complementary to the TpG for unmethylated alleles. A matched oligomer-template DNA molecule hybrid will allow for the incorporation of a labeled nucleotide immediately upstream (5’) to the interrogated CpG (or TpG) site. However, if the probe and template are mismatched, then primer extension will not occur. Adenine and thymine nucleotides are labeled with cy5 (red), while cytosine nucleotides are labeled with cy3 (green). No insertion of guanine nucleotides occurs in Infinium I assays. Of note, the identity of the dye is representative of the nucleotide adjacent to the CpG dinucleotide. The methylation discrimination is derived from separate measurements from the two different types of beads present for each locus. For some loci, both measurements will be cy3, and for others both will be cy5. The Infinium type II chemistry is a true two-color system. A matched oligomer-template DNA molecule hybrid will allow for the incorporation of a labeled nucleotide at the interrogated C or T of the CpG site. Adenine nucleotides labeled with cy5 (red) are incorporated across from unmethylated (TpG) sites, while guanine nucleotides labeled with cy3 (green) are incorporated across from methylated (CpG) sites.
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| Scan protocol |
After the chemistry steps, BeadArrays are scanned and the raw signal intensities are extracted from the *.IDAT files using the ‘noob’ function in the minfi R package.
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| Description |
202878330154_R02C01
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| Data processing |
To classify prospective vestibular schwannomas into molecular groups, a methylation-based classifier using differentially methylated probes from our discovery cohort of 66 tumors was used to construct a support vector machine (SVM) using caret version 6.0 in R version 3.6.0. A linear kernel SVM was constructed using training data comprising 75% of randomly selected samples from the discovery cohort with 10-fold cross validation. The top 2000 differentially methylated probes were used as variables. The model was applied to a test set comprising 25% of randomly selected samples from the discovery cohort, and receiver operating characteristics were acquired for 1000x resampling of test data. Methylation profiles were then processed using this statistical model for molecular group classification.
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| Submission date |
Jan 02, 2023 |
| Last update date |
Jan 02, 2023 |
| Contact name |
John Liu |
| E-mail(s) |
john.liu@ucsf.edu
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| Organization name |
UCSF
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| Street address |
35 Medical Center Way
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| City |
SAN FRANCISCO |
| State/province |
California |
| ZIP/Postal code |
94143 |
| Country |
USA |
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| Platform ID |
GPL21145 |
| Series (1) |
| GSE222042 |
Epigenetic reprogramming shapes the cellular landscape of schwannoma, DNA Methylation |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM6912278_202878330154_R02C01_Grn.idat.gz |
7.0 Mb |
(ftp)(http) |
IDAT |
| GSM6912278_202878330154_R02C01_Red.idat.gz |
7.0 Mb |
(ftp)(http) |
IDAT |
| Processed data not provided for this record |
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