|
| Status |
Public on Nov 11, 2021 |
| Title |
2DT1C2D: LKR10_T1_Cisplatin_Rep4 |
| Sample type |
SRA |
| |
|
| Source name |
LKR10 cell line
|
| Organism |
Mus musculus |
| Characteristics |
tissue: LKR10 cell line
timepoint: T1
replicate: Replicate4
|
| Treatment protocol |
Samples (1e6 cells) were taken for gDNA after passaging once in NGS flanks, and again after re-growing in NGS mice and treating with 3 weekly doses of 7mg/kg cisplatin. In parallel, gDNA was taken from LKR10 cells at identical timepoints before and after cisplatin treatment (10µM cisplatin) for comparison.
|
| Growth protocol |
The murine derived xenograft (MDX) line was created by serial passaging of a single cell suspension of primary tumor cells from the KP mouse model by i.p. injection into the flanks of NOD scid IL2Rgnull (NSG) mice, analogous to PDX generation methods. After growth in NGS mice, primary xenografts were dissociated into single cells and spinfected with the lentiviral shRNA pool,then re-injected directly into NSG mice without selection or attachment on plastic dishes after spinfection. LKR10 cells were grown in DMEM, 10% FBS, and Pen/Strep/Glutamine.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using the Qiagen DNeasy Blood & Tissue kit
Lentiviral pLKO shRNA vectors were amplified using PCR with ExTaq, and each sample was given direct barcodes for multiplexing and Illumina compatible adapters using a secondary PCR step.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
Library strategy: shRNAseq
Original fastq file from the MiSeq was composed of 32 multiplexed samples. Each fastq was split by a barcode identifier using the FASTX-toolkit barcode_splitter.pl script
Count files were created in R (v. 3.1.1) using the edgeR package (v. 3.6.8) processHairpinReads function with no mismatches or shifting allowed, shRNAs with no counts were filtered out and all shRNAs were given an extra count to avoid dividing by zero counts
Analysis of shRNA abundance was performed in edgeR using a time-course design and generalized linear models to compare the initial (T0) and final (T1) timepoints of the untreated cells and the the final timepoints of untreated and cisplatin treated cells and a likelihood ratio test (using the glmLRT function) was used to report log2 fold change scores and significance.
Genome_build: no alignment necessary
Supplementary_files_format_and_content: tab delimited text files containing either counts for each shRNA (*.counts.txt) or log2 fold change and likelihood ratio scores for each shRNA (*Analysis.txt)
|
| |
|
| Submission date |
Aug 26, 2019 |
| Last update date |
Nov 11, 2021 |
| Contact name |
David Simpson |
| E-mail(s) |
David.Simpson@ucsf.edu
|
| Organization name |
UCSF
|
| Street address |
1550 4th Street
|
| City |
San Francisco |
| State/province |
California |
| ZIP/Postal code |
94158 |
| Country |
USA |
| |
|
| Platform ID |
GPL16417 |
| Series (2) |
| GSE136317 |
Identification of Ankrd35 as a mediator of cisplatin response in non-small cell lung cancer by in vivo screening of murine tumor derived xenografts |
| GSE136346 |
Identification of Ankrd35 as a mediator of cisplatin response in non-small cell lung cancer by ex vivo 3D screening of primary tumor spheroids |
|
| Relations |
| BioSample |
SAMN12635600 |
| SRA |
SRX6758729 |
