GSM4826104: CD8SP short-read [CD8SP_L002]; Mus musculus; RNA-Seq
Total RNA was then extracted from the sorted cells with on GenElute Total RNA Purification columns (Sigma). PacBio libraries were constructed following the IsoSeq Express Template Preparation for Sequel Systems protocol (www.pacb.com). For this procedure, first strand cDNA synthesis was performed using SMARTer PCR cDNA Synthesis Kit (Clontech), which also add necessary adaptor sequences for subsequent PCR amplification. PCR amplification of the cDNA was then performed using PrimerSTAR GXL DNA Polymerase (Clontech) and purified using AMPure PB magnetic beads (PacBio). The purified cDNAs were then end-repaired and ligated using the Template Prep Kit (PacBio) to generate the circular SMRTbell libraries. Each library was then sequenced on individual PacBio SMRT cells. For Illumina libraries, the RNA concentration and integrity of samples were first determined with a LabChip GX (Perkin Elmer). Paired-end RNA-seq libraries were then constructed with the TruSeq total RNA sample preparation kit with rRNA-depletion (Illumina) and sequenced on an Illumina NextSeq at the Ramaciotti Centre for Genomics, University of New South Wales.