|
| Status |
Public on Aug 07, 2024 |
| Title |
BT594.T85B.RT-TMZ [GBM_66] |
| Sample type |
SRA |
| |
|
| Source name |
BT594
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: BT594
cell type: primary GBM
treatment: Temozolomide + Radiation (IC20-IC30)
|
| Treatment protocol |
Cells were transduced with the genome-wide TKOv3 'all-in-one' CRISPR-Cas9 library, selected with puromycin, and cultured as indicated.
|
| Growth protocol |
Cells were cultured in Neurocult Complete media supplemented with 1X antibiotic/antimycotic and serially passaged for up to 90 days in biological triplicates. Aliquots were pelleted at indicated timepoints for gDNA extraction and quantification by next-generation sequencing.
|
| Extracted molecule |
other |
| Extraction protocol |
Genomic DNA was extracted using the Wizard Genomic DNA Purification kit (Promega) according to manufacturer’s instructions
For each sample, the gRNA cassette was amplified directly from genomic DNA using primers harboring Illumina TruSeq adaptors with i5 and i7 barcodes. The resulting sequencing libraries were pooled and gel-purified.
|
| |
|
| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
sgRNA amplified from gDNA
|
| Data processing |
FASTQ files were pre-processed to extract Cas9 guide sequence using a bespoke Perl script.
The Cas9 guide sequence was extracted from read1 files as the 20 bases preceeding 'CGGTGTTT'.
Trimmed FASTQ files were then aligned to the TKOv3 FASTA file using bowtie v0.12.1 using the following command line parameters: -p 16 -v 3 -l 18 --chunkmbs 256 -t <library_file> --un unmapped.fastq --al mapped.fastq -1 <trimmed_read1.fastq> aligned.sam
The number of reads aligning to each guide sequence was enumerated for each aligned .sam file, and alignment statistics were recorded. Guide IDs and counts were written to individual count files.
All individual count files were merged along with library annotations in R.
Assembly: hg19
Supplementary files format and content: tab-delimited read count matrix
Library strategy: CRISPRseq
|
| |
|
| Submission date |
Jun 05, 2024 |
| Last update date |
Aug 07, 2024 |
| Contact name |
Jason Moffat |
| E-mail(s) |
jason.moffat@sickkids.ca
|
| Organization name |
Hospital for Sick Children
|
| Department |
Genetics and Genome Biology
|
| Lab |
Moffat
|
| Street address |
686 Bay Street
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G0A4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE240418 |
Functional mapping of Glioblastoma recurrence reveals targetable dependencies in an axonal guidance pathway in lethal brain cancers [Genome-wide CRISPR-Cas9] |
| GSE240492 |
Functional mapping of Glioblastoma recurrence reveals targetable dependencies in an axonal guidance pathway in lethal brain cancers |
|
| Relations |
| BioSample |
SAMN41694253 |
| SRA |
SRX24816259 |
