GSM7140868: CD8+, WT, LGL299; Homo sapiens; RNA-Seq
Purified T-LGL were obtained from PBMC with magnetic micro-beads (Miltenyi Biotec) coated with monoclonal anti-human CD57 or CD56 mAbs, for T-LGLL patients, or CD8 for CTR. Purity and viability of purified cell populations were assessed by flow cytometry. A threshold of at least 98% T-LGLs on all the acquired events, coupled with a contamination lower than 0.1% of monocytes, was accepted. Total RNA was extracted from purified T-LGL with the RNeasy Mini Kit (Qiagen) in combination with on-column DNase I digestion (RNase-Free DNase set, Qiagen), according to manufacturer's instructions. RNA quality was assessed using the RNA 6000 Nano and RNA 6000 PICO assays on a 2100 Bioanalyzer (Agilent). Sequencing libraries were prepared using a TruSeq Stranded Total RNA Kit adapted for long fragments (±550 bp) with the Ribo-Zero Gold rRNA Removal Kit (Illumina) according to the manufacturer's instructions. Prepared libraries were run on a HiSeq3000 high-throughput sequencing system (Illumina) and paired-end reads were generated (315 million reads per sample, on average).