|
| Status |
Public on Nov 04, 2019 |
| Title |
shRNAseq_rep3_DOX_T0 |
| Sample type |
SRA |
| |
|
| Source name |
SW480TetOnAPC cells
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: human colorectal cancer cell line
cell line: SW480TetOnAPC
treatment: DOX
shRNA: Decipher_shRNA_Library_Human_Module_1
|
| Treatment protocol |
SW480TetOnAPC cells were treated with 0.5 µg/ml doxycycline or ethanol.
|
| Growth protocol |
SW480TetOnAPC cells were grown in RPMI 1640 (Sigma). All media were supplemented with 10% FBS (Sigma) and 1% penicillin/streptomycin (Sigma).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from harvested cells using a cell-lysis buffer containing SDS and RNaseA. DNA was sheared by sonification and purified using a phenol-choroform extraction followed by an isopropanol precipitation.
To retrieve Decipher-18bp-barcode-sequences that have previously been integrated together with the shRNA sequences into the genomic host DNA via lentiviral transduction, custom PCR-primers were used that specifically bind to the pRSI9-Decipher vector at sequences flanking the shRNA-specific barcode sequence. In two subsequent PCRs, the barcode sequences were first recovered from the genomic DNA and second relevant adapter-sequences were introduced for hybridization of the PCR-products to the Illumina GAIIx Next-Generation-Sequencer following gel-purification and quantification and size determination using the Experion Automated Electrophoresis System (Bio-Rad).
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
shRNA screen
PCR-product (from genomic DNA)
processed data file:
shRNAseq_counttable.txt
|
| Data processing |
Library strategy: shRNA screen
For shRNA screen analysis, fastq files were mapped to the Decipher-human-module-1 reference database containing all 18-nucleotide barcode-sequences that correspond to the almost 25700 individual shRNA sequences that are contained in the library Decipher_HumanModule1 (Cellecta). This reference sheet is attached as the supplementary file "Reference_barcode_sequences_Decipher_HumanModule1". Mapping was done using the Bacrode-Deconvoluter-Software (The Decipher Project; Cellecta) with default settings.
The counts of aligned reads for all replicates and conditions were combined in a count-table. Enrichment or depletion of shRNAs between two distinct screening-conditions was calculated using the publicly available Galaxy-based shRNAseq tool (edgeR-based statistics).
Genome_build: N/A
Supplementary_files_format_and_content: shRNAseq_counttable.txt: Count matrix containing an identifier that unambiguously identifies a barcode and corresponding shRNA sequence. Furthermore, this count matrix contains the counts of aligned reads for all shRNAs of all samples. This count table was used as input for the analyses with the tool shRNAseq.
|
| |
|
| Submission date |
Jun 15, 2018 |
| Last update date |
Nov 04, 2019 |
| Contact name |
Martin Eilers |
| Organization name |
University of Wuerzburg
|
| Department |
Chair for Biochemistry and Molecular Biology
|
| Lab |
Martin Eilers
|
| Street address |
Am Hubland
|
| City |
Wuerzburg |
| ZIP/Postal code |
97074 |
| Country |
Germany |
| |
|
| Platform ID |
GPL10999 |
| Series (1) |
| GSE106858 |
A MYC/GCN2/eIF2alpha negative feedback loop limits protein synthesis to prevent MYC-dependent apoptosis in colorectal cancer |
|
| Relations |
| BioSample |
SAMN09431492 |
| SRA |
SRX4222761 |
