GSM5328147: PS- CD8 T cells patient1; Homo sapiens; RNA-Seq
Peripheral blood was collected into lithium heparin tubes (Sarstedt). PBMCs were isolated by Biocoll density gradient (Merck) centrifugation and then directly stained for FACS sorting. Live, Dump-, C8+,CCR7+CD45RA-, CCR7-CD45RA-, CCR7-CD45RA+, PS+ and PS- were directly sorted into TRIZOL on a FACSAriaFusion (BD) using a 100 μm nozzle. Total RNA was isolated and purified using Monarch columns (NEB). Poly(A)+ RNA was isolated from the total RNA sample. First-strand cDNA synthesis was primed with a N6 randomized primer. After fragmentation, the Illumina TruSeq sequencing adapters were ligated in a strand specific manner to the 5' and 3' ends of the cDNA fragments. In this way, a strand specific PCR amplification of the cDNA was achieved using a proof-reading enzyme. For Illumina NextSeq sequencing, the samples were pooled in approximately equimolar amounts. The cDNA pool in the size range of 250-700 bp was eluted from a preparative agarose gel. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. The cDNA size range was 250-700 bp. The cDNA pool was sequenced on an Illumina NextSeq 500 system using 1x75 bp read length.