title: RNA-Seq of PPTC PDX models: Sample OS-55-SBX
design: Strand-specific, poly-A+ RNA-seq libraries for sequencing on the Illumina platform were prepared as previously described with minor modifications (Wang et al. 2015; Peters et al. 2015). RNA Integrity was confirmed (RIN >7.0) on a Bioanalyzer (Agilent).  Briefly, poly-A+ mRNA was extracted from 1 Î¼g total RNA using Oligo(dT)25 Dynabeads (Life Technologies), to which 4 ÂµL of 1:100 dilution of the ERCC spike-in mix 1 (Ambion, Life technologies) was already added (Baker et al. 2005). There are a total of 92 poyladenylated transcripts in this mix that are used to monitor sample and process consistency. mRNA is then fragmented by heat at 94 Â°C for 15 minutes or less depending on sample RIN. First strand cDNA was synthesized using NEBNext RNA First Strand Synthesis Module (New England BioLabs) and during second strand cDNA synthesis, dNTP mix containing dUTP was used to introduce strand-specificity with NEBNext Ultra Directional RNA Second Strand Synthesis Module (New England BioLabs). For Illumina paired-end library construction, the resultant cDNA is processed through end-repair and A-tailing, ligated with Illumina PE adapters, and then digested with 10 units of Uracil-DNA Glycosylase (New England BioLabs). Libraries are prepared on the Beckman BioMek FXp robots and amplification of the libraries was performed for 13 PCR cycles using the Phusion High-Fidelity PCR Master Mix (New England BioLabs); 6-bp molecular barcodes that were also incorporated during this step. Libraries were purified with Agencourt AMPure XP beads (Beckman Coulter) after each enzymatic reaction, and after PCR amplification, and were quantified using Fragment Analyzer electrophoresis system. Libraries were pooled in equimolar amounts (4 libraries/pool). Library templates were prepared and s DNA Sequencing.  Sequencing runs generated approximately 300-400 million successful reads on each lane of a flow cell, yielding 75-100M reads per sample.
library_name: OS-55-SBX-HUMAN-RNA
attributes.tag: alignment_software
attributes.value: https://github.com/marislab/pdx-mouse-subtraction