GSM6031099: Perfusate_d9_CD56loCD16+ [000008514669_B04]; Homo sapiens; RNA-Seq
NK cell subpopulations cells were stained with the following antibodies: CD56, clone NCAM16.2; CD16, clone 3G8; CD3, clone UCHT1; CD45, clone HI30 (all BD Biosciences); CD127, clone REA614 (Miltenyi Biotec); with propidium iodide (ThermoFischer Scientific, Massachusetts, USA) for live/dead discrimination. NK cell subpopulations were then sorted to >98% purity using either a FACS Aria or a FACS Melody (BD Biosciences). A total of 1,500 cells of each sub-population of interest was FACS sorted into 1x TCL buffer (Qiagen, Manchester, UK) with 1% b-mercaptoethanol (molecular biology grade, v/v; Sigma-Aldrich, Missouri, USA). Sorted samples were frozen on dry-ice and stored at -80°C until library preparation. Libraries were constructed using a modified version of the SMART-Seq2 protocol (as described in Picelli et. al., 2013 and Picelli et. al., 2014). Polyadenylated transcripts were captured, reverse transcribed, then template switching was performed and pre-amplification used to increase both yield and length of cDNA inserts generated. The generated libraries were sequenced on an Illumina NextSeq 500using a High Output kit to generate 2 x 25 bp indexed reads.