GSM2492556: WT-1; Mus musculus; RNA-Seq
Total RNA was isolated from liver tumors using Trizol (Invitrogen) followed by additional cleanup and DNase digestion using the RNeasy Mini Kit (Qiagen). Four micrograms of total DNase treated RNA were prepared with the TruSeq Stranded Total RNA LT Sample Prep Kit from Illumina. Poly-A RNA is purified and fragmented before strand specific cDNA synthesis.  cDNA were A-tailed and indexed adapters were ligated. Samples were PCR amplified and purified with AmpureXP beads and validated on the Agilent 2100 Bioanalyzer. Before being normalized and pooled, samples were quantified by Qubit (Invitrogen). Sequencing was performed on a Illumina Hiseq 2500 to generate 51-bp single-end reads. Four g of total DNase treated RNA was prepared with the TruSeq Stranded Total RNA LT Sample Prep Kit from Illumina.   Poly-A RNA was purified and fragmented before strand specific cDNA synthesis.  cDNA was A-tailed and indexed adapters were ligated.  Samples were PCR amplified and purified with AmpureXP beads and validated on the Agilent 2100 Bioanalyzer.  Samples were quantified by Qubit (Invitrogen) prior to normalization and pooling.  Sequencing was performed on an Illumina Hiseq 2500 to generate 51-bp single-end reads.  Reads were trimmed to remove low-quality regions in the ends.  Trimmed reads were mapped to the mouse genome (mm10) using TopHat v2.0.1227 guided by iGenomes GTF file (https://ccb.jhu.edu/software/tophat/igenomes.shtml).  Alignments with mapping quality less than 10 were discarded.  Expression abundance estimation and differential expression analysis were carried out using Cufflinks/Cuffdiff (v2.1.1) software.