GSM6523600: mPiC_d2_1; Mus musculus; RNA-Seq
1x10^6 T cells were harvested in RNAprotect Cell Reagent and stored at -80°C o/n before total RNA was extracted using the RNeasy Plus Micro Kit (both Quiagen) RNA quality and quantity were checked using a 2100 Bioanalyzer with the RNA 6000 Nano kit (Agilent Technologies) and RNA Pico kit depending on the sample concentration. The RIN for all samples was >7. cDNA libraries suitable for sequencing were prepared from 5 ng of total RNA with SMART-Seq v4 Ultra Low Input RNA Kit (Takara) according to manufacturer's instructions (1/4 volume). Libraries were quantified by QubitTM 3.0 Fluometer (ThermoFisher) and quality was checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent). 0.5 ng of each library was subjected to a tagmentation-based protocol (Nextera XT, Illumina) using a quarter of the recommended reagent volumes. Libraries were quantified again by QubitTM 3.0 Fluometer (Thermo Fisher) and quality was checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent) before pooling. Sequencing of pooled libraries, spiked with 1% PhiX control library, was performed at 19-36 million reads/sample in single-end mode with 75 nt read length on the NextSeq 500 platform (Illumina) with 1 High Output Kit v2.5. Sequencing of pooled libraries, spiked with 1% PhiX control library, was performed at ~20 million reads/sample in single-end mode with 75 nt read length on the NextSeq 500 platform (Illumina) with 1 High Output Kit v2.5. Demultiplexed FASTQ files were generated with bcl2fastq2 v2.20.0.422 (Illumina).