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Sample GSM4392101 Query DataSets for GSM4392101
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

Supplementary file Size Download File type/resource
GSM4392101_Week19_EGFR_p53_Cas9_vehicle.txt.gz 492.4 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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