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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 04, 2021 |
| Title |
KTC_sg-85-Pool_15Wks |
| Sample type |
SRA |
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| Source name |
Lung epithelial cell
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| Organism |
Mus musculus |
| Characteristics |
viral pool: Lenti-sgTS85/Cre
weeks after tumor initiation: 15
genotype: KT;Cas9
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| Treatment protocol |
Tumors were initiated by intratracheal delivery of 60 µl of lentiviral pools.
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| Growth protocol |
All experiments were performed in accordance with Stanford University Institutional Animal Care and Use Committee guidelines.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from bulk tumour-bearing lung tissue from each mouse. Twelve benchmark control cell lines (3 cell lines of 500,000 cells each, 3 cell lines of 50,000 cells, 3 cell lines of 5,000 cells, and 3 cell lines of 500 cells) were added to each mouse lung sample prior to lysis to enable the calculation of the absolute number of neoplastic cells in each tumour from the number of sgID-BC reads.
Q5 High-Fidelity 2x Master Mix (New England Biolabs, M0494X) was used to amplify the sgID-BC region from 32 µg of genomic DNA using unique dual-indexed primers.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
barcode sequencing from 10 mice
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| Data processing |
Each read is expected to contain an 8-nucleotide sgID region followed by a 30-nucleotide barcode (BC) region (GCNNNNNTANNNNNGCNNNNNTANNNNNGC), and each of the 20 Ns represent random nucleotides with roughly equal representation of A, T, G and C.
The sgID region identifies the putative tumour suppressor gene being targeted, for which we require perfect match between the sequence in the forward read and one of the 102 forward sgIDs with known sequences.
We require the forward and reverse read to agree completely within the 30 nucleotide sequence to be further processed. Any “tumours” that are within a Hamming distance of two from a larger tumour is assigned as “spurious tumours”, which are likely to be resulting from sequencing or PCR error, and are removed from subsequent analysis.
The tumour size (number of neoplastic cells) is calculated by normalizing the number of reads to the three benchmarks “spike-in” cell lines (5x105 cells for each benchmark cell line) added to each sample prior to lysis of the lung and DNA extraction step.
Supplementary_files_format_and_content: Txt files showing the tumor sgRNA, barcode and tumor size for each identified tumor
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| Submission date |
Mar 03, 2020 |
| Last update date |
Feb 04, 2021 |
| Contact name |
Chuan Li |
| E-mail(s) |
chuanli@stanford.edu
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| Organization name |
Stanford University
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| Department |
Biology
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| Lab |
Dmitri Petrov lab
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| Street address |
327 Campus Drive
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE146302 |
A functional taxonomy of tumor suppression in oncogenic KRAS-driven lung cancer |
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| Relations |
| BioSample |
SAMN14274256 |
| SRA |
SRX7842791 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4377001_KTC_sg-85-Pool_15Wks.txt.gz |
385.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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