GSM5944406: 000008512269_H02; Homo sapiens; RNA-Seq
GSM5944406
Naïve (CD45RA+CD28+), EM (CD45RA-CD28-) and TEMRA (CD45RA+CD28-) CD3+CD8+ T cells were FACS sorted (FACSAria, BD Biosciences; purity greater than 95%) from freshly isolated human PBMCs obtained from 6 KT. Cell pellets were resuspended in RLT buffer (Qiagen) containing 1% β-mercaptoethanol before subsequent RNA extraction using a RNeasy Micro Kit according to the manufacturer's instructions (Qiagen). Quality and quantity of the RNA were assessed by spectrometry (Nanodrop) and electrophoresis (Agilent RNA 6000 Pico Kit). Smart-Seq2 libraries were prepared by the Broad Technology Labs and sequenced by the Broad Genomics Platform according to the SmartSeq2 protocol (Picelli et al, 2013, 2014) with some modifications (Trombetta et al, 2014). Briefly, total RNA was purified using RNA-SPRI beads, polyA mRNA was converted to cDNA and amplified cDNA was subject to transposon-based fragmentation that used dual-indexing to barcode each fragment of each converted transcript with a combination of barcodes specific to each sample. Sequencing was carried out as paired-end 2x25bp with an additional 8 cycles for each index.