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Sample GSM4377039 Query DataSets for GSM4377039
Status Public on Feb 04, 2021
Title Ambra1_KPTC
Sample type SRA
 
Source name Lung epithelial cell
Organism Mus musculus
Characteristics viral pool: NL_Ambra1_Palbo_PoolA
weeks after tumor initiation: 15
genotype: KPT;Cas9
Treatment protocol Tumors were initiated by intratracheal delivery of 60 µl of lentiviral pools.
Growth protocol All experiments were performed in accordance with Stanford University Institutional Animal Care and Use Committee guidelines.
Extracted molecule genomic DNA
Extraction protocol Genomic DNA was isolated from bulk tumour-bearing lung tissue from each mouse. Three benchmark control cell lines of 500,000 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.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 2500
 
Description barcode sequencing from 4 mice
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
 
Submission date Mar 03, 2020
Last update date Feb 04, 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)
GSE146303 The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D
Relations
BioSample SAMN14274234
SRA SRX7842829

Supplementary file Size Download File type/resource
GSM4377039_Ambra1_KPTC.txt.gz 972.1 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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