|
| Status |
Public on Apr 30, 2020 |
| Title |
PTCC-2008-P4 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Papillary transitional cell carinoma of the thyroid
|
| Organism |
Homo sapiens |
| Characteristics |
patient diagnosis: Lynch Syndrome
tissue source: Flow sorted tumor from surgical resection
tissue: Papillary transitional cell carinoma of the thyroid
tissue preparation: FFPE
flow sort content: Human tumor
|
| Treatment protocol |
All samples were from treatment naïve surgical resections
|
| Growth protocol |
Formalin fixed paraffin embedded (FFPE) tissue samples were collected at room temperature.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Excess paraffin was removed from each FFPE sample with a scalpel from either side of 40-60 μm scrolls). We used one to three 50 µm scrolls from each FFPE tissue block to obtain sufficient numbers of intact nuclei for subsequent sorting and molecular assays. Biopsies were dewaxed, digested then minced in the presence of NST buffer and DAPI according to published protocols (Holley et al, 2012). Nuclei were disaggregated then filtered through a 40 μm mesh prior to flow sorting with an Influx cytometer (Becton-Dickinson, San Jose, CA) with ultraviolet excitation and DAPI emission collected at >450 nm. DNA content and cell cycle were analyzed using the software program MultiCycle (Phoenix Flow Systems, San Diego, CA). DNAs were extracted using Qiagen micro kits (Qiagen Valencia, CA).
|
| Label |
Cy3
|
| Label protocol |
DNAs from FFPE tissue were treated with DNAse 1 for 1 minute prior to Klenow-based labeling. In each case 1 ul of 10x DNase 1 reaction buffer and 2 μl of DNase 1 dilution buffer were added to 7 μl of DNA sample and incubated at room temperature then transferred to 70°C for 30 minutes to deactivate DNase 1. Sample and reference templates were then labeled with Cy-5 dUTP and Cy-3 dUTP respectively using a BioPrime labeling kit (Invitrogen, Carlsbad, CA) according to our published protocols. All labeling reactions were assessed using a Nanodrop assay (Nanodrop, Wilmington, DE) prior to mixing and hybridization to CGH arrays (Agilent Technologies, Santa Clara, CA).
|
| |
|
| Channel 2 |
| Source name |
Reference Normal female genome
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Reference pooled 46XX
|
| Treatment protocol |
All samples were from treatment naïve surgical resections
|
| Growth protocol |
Formalin fixed paraffin embedded (FFPE) tissue samples were collected at room temperature.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Excess paraffin was removed from each FFPE sample with a scalpel from either side of 40-60 μm scrolls). We used one to three 50 µm scrolls from each FFPE tissue block to obtain sufficient numbers of intact nuclei for subsequent sorting and molecular assays. Biopsies were dewaxed, digested then minced in the presence of NST buffer and DAPI according to published protocols (Holley et al, 2012). Nuclei were disaggregated then filtered through a 40 μm mesh prior to flow sorting with an Influx cytometer (Becton-Dickinson, San Jose, CA) with ultraviolet excitation and DAPI emission collected at >450 nm. DNA content and cell cycle were analyzed using the software program MultiCycle (Phoenix Flow Systems, San Diego, CA). DNAs were extracted using Qiagen micro kits (Qiagen Valencia, CA).
|
| Label |
Cy5
|
| Label protocol |
DNAs from FFPE tissue were treated with DNAse 1 for 1 minute prior to Klenow-based labeling. In each case 1 ul of 10x DNase 1 reaction buffer and 2 μl of DNase 1 dilution buffer were added to 7 μl of DNA sample and incubated at room temperature then transferred to 70°C for 30 minutes to deactivate DNase 1. Sample and reference templates were then labeled with Cy-5 dUTP and Cy-3 dUTP respectively using a BioPrime labeling kit (Invitrogen, Carlsbad, CA) according to our published protocols. All labeling reactions were assessed using a Nanodrop assay (Nanodrop, Wilmington, DE) prior to mixing and hybridization to CGH arrays (Agilent Technologies, Santa Clara, CA).
|
| |
|
| |
| Hybridization protocol |
Labeled DNA was hybridized without ozone scavenger and chips washed according to manufacture's protocol for 40 hours in a rotating 65°C oven.
|
| Scan protocol |
All microarray slides were scanned using an Agilent 2565C DNA scanner and the images were analyzed with Agilent Feature Extraction version 11.0 using default settings.
|
| Data processing |
Data was extracted from the TIFF files using Agilent FE 11. The aCGH data was assessed with a series of QC metrics then analyzed using an aberration detection algorithm (ADM2)[23]. The latter identifies all aberrant intervals in a given sample with consistently high or low log ratios based on the statistical score derived from the average normalized log ratios of all probes in the genomic interval multiplied by the square root of the number of these probes. This score represents the deviation of the average of the normalized log ratios from its expected value of zero and is proportional to the height h (absolute average log ratio) of the genomic interval, and to the square root of the number of probes in the interval.
|
| |
|
| Submission date |
Mar 09, 2020 |
| Last update date |
May 01, 2020 |
| Contact name |
Michael Thomas Barrett |
| E-mail(s) |
barrett.michael@mayo.edu
|
| Phone |
480-301-6736
|
| Organization name |
Mayo Clinic Arizona
|
| Department |
Molecular Pharmacology and Experimental Therapeutics
|
| Street address |
13400 East Shea Boulevard
|
| City |
Scottsdale |
| State/province |
AZ |
| ZIP/Postal code |
85259 |
| Country |
USA |
| |
|
| Platform ID |
GPL19387 |
| Series (1) |
| GSE146670 |
Unique genomic and neoepitope landscapes across tumors: a study across time, tissues, and space within a single Lynch Syndrome patient |
|
