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Sample GSM3941271

Query DataSets for GSM3941271

Status

Public on Mar 10, 2020

Title

A20I-405225-T-RNA [S105]

Sample type

SRA

Source name

Allograft_20Gy_Iso_Tumor

Organism

Mus musculus

Characteristics

strain background: 129/SvJae
replicate group: Allograft_20Gy_Iso_Tumor_RNA
barcode: GAATTCGT+ATAGAGGC
tumor type: Allograft
radiation dose: 20Gy
treatment: Isotype

Treatment protocol

Tumors were treated when volume reached ~150 mm3 and harvested 3 days later. Mice received 200 ug IP of either muDX400 (Merck, murine anti-PD-1) or isotype control followed immediately by 0 or 20 Gy radiation. For mice receiving radiation, the tumor-bearing hind limb of the mice was irradiated with 20 Gy using the X-RAD 225Cx small animal image-guided irradiator (Precision X-Ray). The irradiation field included the whole left hind limb and was defined using fluoroscopy with 40 kVp, 2.5 mA X-rays using a 2 mm Al filter. Irradiations were performed using parallel-opposed anterior and posterior fields with an average dose rate of 300 cGy/min prescribed to midplane with 225 kVp, 13 mA X-rays using a 0.3 mm Cu filter.

Growth protocol

Primary p53/MCA sarcomas were generated in mice between 6-10 weeks old by intramuscular Adeno-Cre injection into the gastrocnemius of p53FL/FL mice, followed by injection of 300 µg MCA (Sigma-Aldrich) resuspended in sesame oil (SigmaAldrich) at 6 µg/µl. Allograft p53/MCA sarcomas were generated by injecting 50,000 cells resuspended in 100 uL DMEM (Gibco) and Corning matrigel from cell line 402230 into the gastrocnemius muscle of syngeneic mice.

Extracted molecule

total RNA

Extraction protocol

Tumor specimens and matched liver control were harvested and stored in RNALater at -80° until all samples were collected. DNA and RNA extractions from each sample were performed using AllPrep DNA/RNA Mini Kit (Qiagen). Extracted total RNA quality and concentration was assessed on a 2100 Bioanalyzer (Agilent Technologies) and Qubit 2.0 (ThermoFisher Scientific), respectively. Only extracts with RNA Integrity Number (RIN) greater than 7 were processed for sequencing. DNA extraction was followed by RNAse treatment (Qiagen). Genomic DNA samples were quantified using fluorometric quantitation on the Qubit 2.0 (ThermoFisher Scientific). For each sample, 200 ng of DNA was sheared using focused-ultrasonicators (Covaris) to generate DNA fragments of about 300 bp in length.
RNA-seq libraries were prepared using the commercially available KAPA Stranded mRNA-Seq Kit (Roche) following the manufacturer’s protocol. In brief, mRNA transcripts were first captured using magnetic oligo-dT beads, fragmented using heat and magnesium, and reverse transcribed using random priming. During the 2nd strand synthesis, the cDNA:RNA hybrid was converted into to double-stranded cDNA (dscDNA) and dUTP incorporated into the 2nd cDNA strand, effectively marking the second strand. Illumina sequencing adapters were then ligated to the dscDNA fragments and amplified to produce the final RNA-seq library. The strand marked with dUTP was not amplified, allowing strand-specificity sequencing.
Libraries were indexed using a dual indexing approach allowing for multiple libraries to be pooled and sequenced on the same sequencing Illumina sequencing flow cell.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

4654-S105_S9_L001

Data processing

Base-calling was done on the instrument using RTA v3.3.3.
Demultiplexing was done using bcl2fatq v2.20.0.422
Sequenced reads were trimmed for adaptor sequence and low-quality sequence using Trimmomatic v0.36
Reads were mapped to mouse mm10 genome using STAR v5.5.4b and quantified to features using HT-Seq built in the STAR aligner.
Genome_build: mm10
Supplementary_files_format_and_content: csv file with read counts per gene

Submission date

Jul 15, 2019

Last update date

Apr 17, 2020

Contact name

David Kirsch

E-mail(s)

david.kirsch@duke.edu

Organization name

Duke University

Street address

PO Box 96100