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Sample GSM3676386

Query DataSets for GSM3676386

Status

Public on Oct 25, 2019

Title

Sx8_1TB

Sample type

SRA

Source name

SKH-1 Mouse

Organism

Mus musculus

Characteristics

strain: SKH-1
infection status: Uninfected
tissue: Skin Tumor

Treatment protocol

Mice were either infected with MmuPV1 or sham infected then completed a 25 week DMBA-UV skin cancer induction protocol. Three weeks following infection, mice were treated on their back skin with 50 ug of DMBA, rested for one week, then subjected to 3 doses of 100 mJ/cm^2 per week for 25 weeks.

Extracted molecule

total RNA

Extraction protocol

Tissue was extacted using the Qiagen RNeasy Mini Kit (50) Cat no. 74104
Novogene: "Briefly, mRNA from Eukaryote organisms is purified from total RNA using poly-T oligo-attached magnetic beads. The mRNA is first fragmented randomly by addition of fragmentation buffer. Then first strand cDNA is synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis is subsequently performed using DNA Polymerase I and RNase H. Double-stranded cDNA is purified using AMPure XP beads. Remaining overhangs of the purified double-stranded cDNA are converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure is ligated to prepare for hybridization(1). In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments are purified with AMPure XP system (Beckman Coulter, Beverly, USA). Finally, the final library is gotten by PCR amplification and purification of PCR products by AMPure XP beads."

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Sham infection, completed 25 wk DMBA-UV Protocol

Data processing

Novogene: Raw image data file from high-throughput sequencing(like Illumina ) was transformed to Sequenced Reads(called Raw Data or Raw Reads) by CASAVA base recognition (Base Calling).
Novogene: "The Sequenced Reads/raw reads often contain low quality reads or reads with adaptors. In order to avoid this, it's necessary to filter the raw reads under conditions to get the clean reads. Raw reads filtering conditions are as follows: (1) Remove reads containing adaptors; (2) Remove reads containing N > 10% (N represents base that could not be determined); (3) The Qscore (Quality value) of over 50% bases of the read is <= 5."
Novogene: "Mapping the clean reads to the reference genome or the transcriptome is the basis for the next following analysis. Novogene use STAR to accomplish the mapping. In order to avoid losing a lot of effective junction reads, Novogene use STAR software to alignment for RNA-seq sequencing data analysis, the junction reads can be positioned precisely."
Novogene: "For the samples with biological replicates, differential expression analysis of two conditions/groups was performed using the DESeq2 R package (Anders et al., 2010). It provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. So, if the readcount of the i-th gene in j-th sample is Kij, there is: Kij ～ NB(μij,σij2). And the resulting P values were adjusted using the Benjamini and Hochberg's approach for controlling the false discovery rate."
Novogene: "Volcano diagram can visually show the whole distribution of differential expression genes. For the samples with biological replicates, the threshold of differential expression genes is: padj < 0.05. For the samples without biological replicates, the threshold of differential expression genes is: |log2(FoldChange)| > 1 and qvalue < 0.005."
Novogene: "Cluster Analysis of differential expression genes was used to estimate expression pattern of differential expression genes under different experimental conditions. Through clustering genes in similar expression pattern, unknown functions of transcripts were recognized, with the reason that same kind of transcripts have similar functions or participate in the same metabolic processes or cellular pathways. Hierarchical clustering analysis was carried out with the log10(FPKM+1) of union differential expression genes of all comparison groups under different experimental conditions."
Genome_build: mm10
Supplementary_files_format_and_content: readcount_genename.txt - contains read counts for all genes across all samples. Used for subsequent analysis performed by Novogene.

Submission date

Mar 18, 2019

Last update date

Oct 25, 2019

Contact name

Jonathan Leigh Messerschmidt

E-mail(s)

jmesserschmidt@mgh.harvard.edu

Organization name

Massachusetts General Hospital

Department

Center for Cancer Immunology, Cutaneous Biology Research Center, Center for Cancer Research