RNA-Seq Protocol for CD4+ T Cells

CD4+ T cells were isolated using negative selection (Stem Cell Technologies CD4 enrichment kit) according to the manufacturer’s recommendations. Total RNA was extracted using the Qiagen RNA Kit with Qiashredder columns. Material was quantified using RiboGreen (Invitrogen) on the FLUOstar OPTIMA plate reader (BMG Labtech) and the size and integrity analysed on the 2200 TapeStation (Agilent, RNA ScreenTape). Input material was normalised to 100 ng prior to library preparation. Polyadenylated transcript enrichment and strand specific library preparation was completed using NEBNext Ultra II mRNA kit (NEB) following manufacturer’s instructions. Libraries were amplified on a Tetrad (Bio-Rad) using in-house unique dual indexing primers^{43 (link)}. Individual libraries were normalised using Qubit, and the size profile analysed on the 2200 TapeStation. Sequencing was performed using an Illumina Novaseq6000 platform at 150 paired end mode (Illumina, San Diego, CA).