GSM9610062: Mali, CD56dim, Siglec-7neg, sort4; Homo sapiens; RNA-Seq
To ensure that each sample had enough NK cells for library preparation and sequencing, equal numbers of thawed PBMCs from three individuals from the same location (either Mali or USA) were combined to generate a sample, and four samples were generated for each location. (A total of 12 individuals were used to generate the four sequencing samples from each location.) NK cells were isolated from each of the combined samples by staining with with Live/Dead, Annexin V, CD64, CD56, CD3, CD7 and Siglec-7, followed by sorting on Live, Annexin V negative, CD64 negative, CD3 negative, CD7+. They were then then further sorted into three populations: CD56dim Siglec-7+, CD56dim Siglec-7neg, CD56neg Siglec-7neg. The cells were sorted into microtubes containing 1x PBS with 50% FBS. Total RNA was then extracted from the cells using the RNeasy Mini Kit  (Qiagen) per the manufacturer's instructions (Qiagen). The purity of RNA was assessed by calculating the ratio of OD260/OD280. The RNA was frozen at -80°C before being sent for sequencing. Libraries were prepped using the SMARTer Stranded Total RNA-Seq Kit V3 – Pico Input Mammalian kit (Takara) following manufacturer's protocols, and sequenced on the Illumina Nova X Plus 10B next-generation sequencer.