4017_WC_5
2 million PBMC were lysed using QIAzol Lysis Reagent (Qiagen). Samples were stored at -80C until RNA extraction. RNA was extracted using the miRNeasy Mini Kit (Qiagen) including DNase treatment according to the manufacturer's instructions. The Smart-seq2 protocol was used to perform full-length bulkRNAseq. Briefly, RNA was captured using oligo-poly(dT)-30 primers and reverse transcription was performed using 50-template switching oligos (LNA technologies, Exicon). cDNA was pre-amplified using PCR and purified twice by magnetic beads (volume ratio 0.6x and 0.8x Ampure-XP magnetic beads, Beckman Coulter). cDNA was measured using capillary electrophoresis (Fragment analyzer, Advance analytical) and 0.5 ng of cDNA was used to generate indexed Illumina libraries (Nextera XT library preparation kit, Illumina). Each library's fragment size was measured by capillary electrophoresis (Fragment analyzer, Advance analytical) and was quantified (Picogreen, Thermofisher). Libraries were pooled at equal molar concentration and sequenced paired-end to a minimum depth of 15 million reads with each a length of 100 base pairs.