|
| Status |
Public on May 01, 2019 |
| Title |
HCT116 wt 3 |
| Sample type |
RNA |
| |
|
| Source name |
HCT116
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
tissue: colorectal carcinoma
|
| Treatment protocol |
No treatment was applied.
|
| Growth protocol |
All cell lines HCT116 and DAPK1 ko clones 7/6, 10/8 and 21/9 were cultured in a 100 cm cell culture dish and maintained in RPMI medium supplemented with 1% penicillin/streptomycin and 10% FBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell harvesting was performed on ice. Cells were scraped off from the cell culture plates and cell pellets were prepared by centrifugation at 5000 rpm fr 5 min at 4 °C. Cells were washed with PBS, centrifugation was repeated, supernatants were discarded and dry cell pellets were frozen in liquid nitrogen and stred at -80°C. RNA was isolated from frozen cell pellets using the QIAzol Lysis Reagent (Qiagen) and RNeasy Mini Kit (Qiagen) according to the manufacturer's protocols.
|
| Label |
Reporter CodeSet & Capture ProbeSet (PanCancer Progression Panel, Nanostring)
|
| Label protocol |
100 ng (contaied in 5 µl) of total RNA per sample was labeled with the Reporter CodeSet & Capture ProbeSet included in the following kit: XT_PGX_HuV1_CancerProg_CSO XT-CSO-PROG1-12(Cat. no. 115000152, Nanostring).
|
| |
|
| Hybridization protocol |
The Nanostring hybridization reaction containing 3 µl Reporter CodeSet, 5 µl hybridization buffer, 2 µl Capture ProbeSet and 5 µl total RNA (100 ng) was conducted for 16 h at 65°C. After hybridization, labelled RNA was processed automatedly according to the manufacturer's instructions for approximately 2.5 to 3 h through the NanoString nCounter Prep Station, where mRNA specific magnetic beads separation and mRNA immobilization on the cartridge surface takes place.
|
| Scan protocol |
Automated counting and tabulating of signals of the reporter probes was performed using the nCounter Digital Analyzer (Nanostring), a multi-channel epifluorescence scanner specifically configured for use with Nanostring's nCounter cartridges, and standard settings.
|
| Description |
Wildtype cancer cell line HCT116
|
| Data processing |
Data were processed internally using the nCounter Digital Analyzer (Nanostring). The number of mRNA specific reporter signals counted corresponds to number of respective mRNA molecules counted and present in the sample. For each sample, one Reported Code Count (RCC) file is generated, which contains the gene expression raw data. RCC files were downloaded from the nCounter Digital Analyzer and imported into the nSolver Analysis Software 3.0 (Nanostring) for data processing, quality control and analysis. Finally, housekeeping gene (geometric mean of 40 genes) normalization for quantitating gene expression levels, positive control normalization for background noise correction and data analysis was performed using nSolver™ Analysis Software 3.0 (NanoString Technologies, Hamburg, Germany) and standard settings. The fold-change of counts was determined by averaging results per DAPK1 ko cell line and comparing them to average counts of HCT116 cells.
Matrix tables contain normalized counts, raw data and fold change (FC) data of nine samples (see samples list above) obtained from the nSolver Analysis Software 3.0. Fold changes of counts were determined by averaging results per DAPK1 ko cell line (n = 1-3) and by comparing them to the average counts of HCT116 cells (n = 3) and are given in excel sheet Grouped FC all ko clones. Two further sheets present individual fold change data of all replicates (HCT116: n = 3, clone 7/6: n = 2, clone 10/8: n = 3, clone 21/9: n=1) and fold changes grouped per ko clone.
|
| |
|
| Submission date |
Apr 30, 2019 |
| Last update date |
May 01, 2019 |
| Contact name |
Regine Schneider-Stock |
| E-mail(s) |
regine.schneider-stock@uk-erlangen.de
|
| Organization name |
University Hospital Erlangen
|
| Department |
Department of Pathology
|
| Lab |
Experimental Tumorpathology
|
| Street address |
Universitaetsstrasse 22
|
| City |
Erlangen |
| State/province |
Bavaria |
| ZIP/Postal code |
91054 |
| Country |
Germany |
| |
|
| Platform ID |
GPL26599 |
| Series (1) |
| GSE130488 |
Comparison of endogenous gene expression of HCT116 colorectal cancer cells and HCT116-derived DAPK1 knockout (ko) clones. |
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