|
| Status |
Public on Feb 15, 2016 |
| Title |
GSC-0131 Day 23 Rep 1 |
| Sample type |
SRA |
| |
|
| Source name |
GBM stem-like cells
|
| Organism |
Homo sapiens |
| Characteristics |
isolate: GSC-0131
time: Day 23
cell type: GBM stem-like cells
source: Primary patient-derived isolates
age: Adult
gbm subtype: Mesenchymal
|
| Treatment protocol |
For large-scale transduction, ~ 220 million GSC or NSC cells were plated into T225 flasks at an appropriate density per biological replicate. Each replicate was transduced such that ~500 fold representation of the library was achieved (at MOI~1 and 30% infection efficiency). 2 days after transduction, puromycin was added (1-4μg/ml) and maintained for 3 days. A portion of cells were harvested as Day 0 time point. The remaining cells were then passaged into T225 flasks maintaining 500 fold representation and cultured for an additional 21-23 days or ~8-10 cell doublings.
|
| Growth protocol |
GSC and NSC lines were grown in N2B27 neural basal media (StemCell Technologies) supplemented with EGF and FGF-2 (20ng/mL each) (Peprotech) on laminin (Sigma) coated polystyrene plates and passaged as previously described. Cells were detached from their plates using Accutase (Millipore). Cells were split every 3-4 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using QiaAmp blood purification Midi kit (Qiagen).
A two-step PCR procedure was employed to amplify sgRNA sequences (first PCR:12 cycles) and then to incorporate deep sequencing primer sites onto sgRNA amplicons (second PCR:20 cycles). For the first PCR, the amount of genomic DNA for each sample was calculated in order to achieve 500-fold coverage over the library (~6.6 μg of gDNA for 106 cells), which resulted in ~213 μg DNA per sample. For each sample, ~100 separate PCR reactions were performed with 2 μg genomic DNA in each reaction using Herculase II Fusion DNA Polymerase (Agilent). Afterwards, a second PCR was performed to add on Illumina adaptors and to barcode samples, using 5ul of the product from the first PCR. We used a primer set to include both a variable 1-6 bp sequence to increase library complexity and 6 bp Illumina barcodes for multiplexing of different biological samples. Resulting amplicons from the second PCR were column purified using the combination of PureLink PCR purification kit (Life Technologies) and MinElute PCR purification kit (Qiagen) to remove genomic DNA and first round PCR product. Purified products were quantified, and mixed.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
sgRNA
131231
|
| Data processing |
Base calling was performed using Illumina's RTA software, v1.17.21.3
Bcl to fastq conversion was conducted using Illumina's CASAVA v1.8.2
Demultiplexing of in line indices was performed using fastx-toolkit
Reads were aligned to the GECKO v1 human sgRNA reference via bowtie 1.0.0, allowing for 0 mismatches
Normalization and significance testing were done using the Bioconductor package edgeR
Genome_build: GECKO v1 human sgRNAs
Supplementary_files_format_and_content: tab delimited text file with annotation and raw counts for each sgRNA across all samples
|
| |
|
| Submission date |
Jun 19, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Patrick Paddison |
| E-mail(s) |
paddison@fhcrc.org
|
| Phone |
206-667-4474
|
| Organization name |
Fred Hutchinson Cancer Research Center
|
| Street address |
1100 Fairview Ave N, C3-187
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE70038 |
Genome-wide CRISPR-Cas9 screens reveal loss of redundancy between PKMYT1 and WEE1 in Glioblastoma stem-like cells |
|
| Relations |
| BioSample |
SAMN03782985 |
| SRA |
SRX1066464 |
