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Sample GSM4239974 Query DataSets for GSM4239974
Status Public on Jan 20, 2020
Title JS181204
Sample type SRA
 
Source name Mouse lung adenocarcinoma (K-rasLA2-G12D/+; p53LSL/LSL; Rosa26-CreERT2+, KPR) cells
Organism Mus musculus
Characteristics cell type: lung adenocarcinoma (K-rasLA2-G12D/+; p53LSL/LSL; Rosa26-CreERT2+, KPR) cells
infected with: sgRNA targeting the Pvt1 p53RE (gΔRE)
treatment: -Tamoxifen
s4u feed: Yes
Treatment protocol Tamoxifen-treated cells had media spiked to a final concentration of 0.5 µM tamoxifen and untreated (no tamoxifen) cells had media spiked with vehicle control. s4U-treated cells had their media spiked with s4U to a final concentration of 100 µM for total RNA-seq or 1 mM for transient-transcriptome RNA-seq. Untreated (no s4U) cells had their media left unsupplemented. Cells were incubated with s4U for 1h or 5m for total RNA-seq or transient-transcriptome RNA-seq, respectively.
Growth protocol KPR cells, previously infected with with an sgRNA targeting the PVT1 p53RE or a nontargeting control, were grown in Dulbecco’s Modified Essential Media supplemented with 10% fetal bovine serum (Gibco), 0.1 mM nonessential amino acids, 2 mM L-glutamine, and Pen/Strep (50 U/ml). All cell cultures were maintained at 37℃ in a humidified incubator with 5% carbon dioxide.
Extracted molecule total RNA
Extraction protocol RNA was harvested with Trizol reagent, followed by chloroform extraction and ethanol precipitation. Genomic DNA was depleted using Turbo DNAse, followed by cleanup with RNAclean beads. RNA for transient-transciptome RNA-seq was enriched with MTS-biotin and all RNA was treated with oxidative-nucleophilic-aromatic-substitution chemistry as described in Schofield et al. Nature Methods 2018.
Libraries were generated following standard protocols using the SMARTer Stranded Total RNA-Seq Kit-Pico Input Mammalian, V2 (Takara Bio USA, cat. 634413).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description Reads filtered for number of T-to-C mutations (0-5+) and strandedness (min/pos)
Data processing Reads were filtered for unique sequences using FastUniq
Reads were trimmed using cutadapt (--minimum-length=20)
Reads were aligned to the GRCm38 genome using HISAT2 aligned with default parameters and --mp 4,2 .
Reads were processed using Picard tools, including FixMateInformation, SortSam and BuildBamIndex
Reads were filtered for those that aligned uniquely (flag: 83/163, 99/147), with MAPQ ≥ 2, and without insertions.
HTSeq-count was used to identify mapped reads in UCSC transcripts (union mode)
T-to-C mutations were identified using Rsamtools and a custom R script (available upon request). Only mutations at positions with a base quality score of greater than 45 that were at least three nt from the end of the read were counted. Reads were excluded where there were greater than five T-to-C mutations and these mutations did not account for at least one third of the observed mutations (NM tag).
SNP sites were filtered from our data in two ways: 1, bcftools was used to identify T-to-C SNP sites in control samples, and these sites were excluded from analysis; 2, sites exhibiting high T-to-C mutation rates in non-s4U treated controls were excluded from analysis.
To examine the distribution of reads with each minimum number of T-to-C mutations, bam files were filtered using Picard tools.
STAR aligner was used to make genome-coverage tracks (inputAlignmentsFromBam mode, outWigType bedGraph), and tracks were normalized using DESeq2 (estimateSizeFactors).
Tracks were converted to binary format using IGVtools (toTDF).
Genome_build: GRCm38
Supplementary_files_format_and_content: bedgraph
 
Submission date Dec 30, 2019
Last update date Jan 21, 2020
Contact name Matthew Simon
E-mail(s) matthew.simon@yale.edu
Organization name Yale University
Department MBB
Lab Simon Lab
Street address West Campus, 100 West Campus Drive, Ste MIC312A
City Orange
State/province CT
ZIP/Postal code 06477
Country USA
 
Platform ID GPL24247
Series (2)
GSE142772 p53 activates the long noncoding RNA Pvt1b to inhibit Myc and suppress tumorigenesis
GSE143204 p53 activates the long noncoding RNA Pvt1b to inhibit Myc and suppress tumorigenesis
Relations
BioSample SAMN13701944
SRA SRX7480745

Supplementary file Size Download File type/resource
GSM4239974_JS181204.TC.0.min.bedGraph.gz 51.1 Mb (ftp)(http) BEDGRAPH
GSM4239974_JS181204.TC.0.pos.bedGraph.gz 53.5 Mb (ftp)(http) BEDGRAPH
GSM4239974_JS181204.TC.1.min.bedGraph.gz 22.7 Mb (ftp)(http) BEDGRAPH
GSM4239974_JS181204.TC.1.pos.bedGraph.gz 24.3 Mb (ftp)(http) BEDGRAPH
GSM4239974_JS181204.TC.2.min.bedGraph.gz 10.2 Mb (ftp)(http) BEDGRAPH
GSM4239974_JS181204.TC.2.pos.bedGraph.gz 11.1 Mb (ftp)(http) BEDGRAPH
GSM4239974_JS181204.TC.3.min.bedGraph.gz 4.0 Mb (ftp)(http) BEDGRAPH
GSM4239974_JS181204.TC.3.pos.bedGraph.gz 4.5 Mb (ftp)(http) BEDGRAPH
GSM4239974_JS181204.TC.4.min.bedGraph.gz 1.4 Mb (ftp)(http) BEDGRAPH
GSM4239974_JS181204.TC.4.pos.bedGraph.gz 1.6 Mb (ftp)(http) BEDGRAPH
GSM4239974_JS181204.TC.5.min.bedGraph.gz 448.7 Kb (ftp)(http) BEDGRAPH
GSM4239974_JS181204.TC.5.pos.bedGraph.gz 522.7 Kb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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