title: RNA-seq of human memory T-cells
design: From 0.5  1 x 10 6 snap-frozen cells total RNA was isolated for mRNA seq and DNaseI treated using the Direct-zol kit (Zymo Research) according to the manufacturers protocol. From ca. 200 ng total RNA, mRNA was bound to streptavidin coated tubes with the mRNA Capture kit (Roche). After three washes, first strand synthesis was performed with 200U M-MLV Reverse Transcriptase (Promega) at 37C for 1.5 h. Second strands were generated using 10U DNA Polymerase I (Thermo Fisher Scientific), 200U T4-Ligase (NEB), 5U RNase H at 16 C for 2.5 h. Then cDNA was tagmented with 0.2 l Tn5 enzyme from the Nextera Library Preparation kit (Illumina) for 5 min at 55 C. After clean-up with the MinElute PCR purification kit (Qiagen) and PCR amplification (9 - 12 cycles) with NEB Next PCR Master mix, samples were purified with AMPure XP beads (Beckman Coulter) and sequenced for 100 basepairs using a V3 single read flow cell on a HiSeq 2500 (Illumina).