GSM2978491: OVA_24h Rep2; Mus musculus; RNA-Seq
Cells were stimulated in vitro as above, and at the times indicated T cells were isolated via immunomagnetic negative selection (StemCell, Vancouver, Canada 19853), RNA was collected as per the manufacturers instruction (Direct-zol RNA MiniPrep Plus w/ TRI Reagent, Zymo Research), treated with DNAse (TURBO DNA-free Kit, Invitrogen), and stored at -80°C until analysis at the UW-Madison Biotechnology Center. Total RNA was verified for purity and integrity via a NanoDrop2000 Spectrophotometer and Agilent 2100 BioAnalyzer, respectively. Samples that met the Illumina sample input guidelines were prepared using the Illumina® TruSeq® Stranded mRNA Sample Preparation kits (Illumina Inc., San Diego, California, USA). For each library preparation, mRNA was purified from 1000 ng total RNA using poly-T oligo-attached magnetic beads. Subsequently, each poly-A enriched sample was fragmented using divalent cations under elevated temperature. The fragmented RNA was synthesized into double-stranded cDNA using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, California, USA) and random primers for first strand cDNA synthesis followed by second strand synthesis using DNA Polymerase I and RNAse H for removal of mRNA. Double-stranded cDNA was purified by paramagnetic beads (Agencourt AMPure XP beads, Beckman Coulter). The cDNA products were incubated with Klenow DNA Polymerase and DNA fragments, ligated to Illumina adapters, and purified by paramagnetic beads. Adapter ligated DNA was amplified in a Linker Mediated PCR reaction (LM-PCR) for 11 cycles using PhusionTM DNA Polymerase and Illumina's PE genomic DNA primer set and then purified by paramagnetic beads. Quality and quantity of the finished libraries were assessed using an Agilent DNA1000 chip (Agilent Technologies, Inc., Santa Clara, CA, USA) and Qubit® dsDNA HS Assay Kit (Invitrogen, Carlsbad, California, USA), respectively. Libraries were standardized to 2μM. Cluster generation was performed using standard Cluster Kit (v3) and the Illumina Cluster Station. Single 100bp sequencing was performed, using standard SBS chemistry (v4) on an Illumina HiSeq2500 sequencer. Images were analyzed using the standard Illumina Pipeline, version 1.8.2. RNA libraries were prepared for sequencing using standard Illumina protocols