|
Status |
Public on Feb 01, 2021 |
Title |
Week19_EGFR;p53;Cas9_vehicle |
Sample type |
SRA |
|
|
Source name |
Lung epithelial cell
|
Organism |
Mus musculus |
Characteristics |
viral pool: Lenti-sgTSPool/Cre weeks after tumor initiation: 19 genotype: EGFR;p53;Cas9 untreated: Vehicle-treated tumors
|
Treatment protocol |
For analysis of tumor growth 11 weeks after tumor initiation, the Lenti-sgTSPool/Cre titer administered to EGFR;p53 mice was 2x10^6 infectious units (ifu)/mouse, while for EGFR;p53;Cas9 mice we used 1x10^6 ifu/mouse. For the 19-week time-point in EGFR;p53;Cas9 mice we initiated tumors with 1x10^5 ifu/mouse. Two weeks before collection, EGFR;p53;Cas9 mice were treated with either vehicle or osimertinib. For Kras;p53;Cas9 mice, we used 2.2x10^5 ifu/mouse.
|
Growth protocol |
All procedures were performed in accordance with protocols approved by the Yale University IACUC and in agreement with the NIH Guide for the Care and Use of Laboratory Animals.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA was isolated from bulk tumour-bearing lung tissue from each mouse. Three cell lines with a defined sgID-BC added at 5x10^5 cell/sample 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 was used to amplify the sgID-BC region from 32 µg of genomic DNA using unique dual-indexed primers.
|
|
|
Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2500 |
|
|
Data processing |
Each read is expected to contain an 8-nucleotide sgID region followed by a 23-nucleotide barcode (BC) region (AANNNNNTTNNNNNAANNNNNAT), and each of the 15 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 15 sgIDs with known sequences. We require the forward and reverse read to agree completely within the 23 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 (5x10^5 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.
|
|
|
Submission date |
Mar 06, 2020 |
Last update date |
Feb 02, 2021 |
Contact name |
Chuan Li |
E-mail(s) |
chuanli@stanford.edu
|
Organization name |
Stanford University
|
Department |
Biology
|
Lab |
Dmitri Petrov lab
|
Street address |
327 Campus Drive
|
City |
Stanford |
State/province |
CA |
ZIP/Postal code |
94305 |
Country |
USA |
|
|
Platform ID |
GPL17021 |
Series (1) |
GSE146550 |
Genetic determinants of EGFR-Driven Lung Cancer Growth and Therapeutic Response In Vivo |
|
Relations |
BioSample |
SAMN14318493 |
SRA |
SRX7862682 |