GSM8966635: 21_IL12Rb2_antiCD3_28_4hr; Homo sapiens; RNA-Seq
Human memory T cells were transduced with lentiviral vectors encoding either IL-23R or IL-12Rβ2. Following transduction, IL-23R⁺ or IL-12Rβ2⁺ cells were isolated and stimulated with IL-23 or IL-12, respectively, for 4 or 12 hours, in the presence or absence of anti-CD3/CD28 co-stimulation. Cells were subsequently harvested for RNA-seq analysis. Twenty-eight RNA samples derived from T cells were provided for this study. Each RNA sample was quantitated by spectrophotometry (NanoDrop, ND-1000, ThermoScientific, DE) and fluorometry (Quant-iT™ PicoGreen® dsDNA reagent,Cat # P7589, Invitrogen, Eugene, OR).  To check for quality, RNA samples were run using RNA6000 Nano Kit (Cat# 5067-1511, Agilent Technologies, Waldbronn, Germany) on an Agilent 2100 Bioanalyzer.  Illumina fragment libraries were constructed using Illumina TruSeq Stranded mRNA kits and protocols (Illumina, Hayward CA).  A unique adapter index sequence, provided in the kit, was used for each sample.  Samples were checked for accurate library sizing using Agilent High Sensitivity DNA Kit (Cat# 5067-4626, Agilent Technologies, Waldbronn, Germany) on an Agilent 2100 Bioanalyzer.  To accurately quantitate the library, qPCR was performed using Kapa Library Quantification Kit for Illumina (KK4835, KapaBioSystems, Boston, MA) on the Quantstudio 12k Flex real-time PCR instrument (Life Technologies, Carlsbad, CA).  Each library was quantitated in triplicate. Samples were diluted to a final dilution of 3 nM, normalized and pooled into 6 separate pools containing 4 or 5 samples each.  Libraries were diluted to a final dilution on average of 10 pM. To check for proper cluster density and sample normalization, a MiSeq single plex 1 x 26 bp +6 bp index sequencing was performed using the MiSeq instrument.  Flow cell clustering was performed using TruSeq PE Cluster Kit v2 (Illumina, Hayward CA) on the Illumina cBot to prepare for HiSeq sequencing. The Illumina HiSeq 2500 sequencer instrument, reagents, and flow cell (Illumina, Hayward CA) were prepared according to Illumina TruSeq PE SBS Kit v5 protocols. Sequencing was performed in paired-end multiplex 2 x 100 bp mode. Basecalling and demultiplexing were performed on the HiSeq 2500 sequencing system (Illumina) using Illumina's Real Time Analysis (RTA) v1.18.61 program.