title: NextSeq 500 paired end sequencing; Blood RNA sequencing in HIV-negative healthy volunteers and people living with HIV on effective, long-term anti-retroviral therapy
design: Blood RNA sequencing in HIV-negative healthy volunteers and people living with HIV on effective, long-term anti-retroviral therapy
library_construction_protocol: Samples were collected as part of a prospective cohort study of people living with HIV attending routine ambulatory HIV care at the Royal Free London NHS Foundation Trust, UK, and of HIV-negative healthy controls recruited from Sexual Health and General Practice clinics. To recruit controls with demographic characteristics similar to the group of people living with HIV, electronic medical records were used to identify participants with age, gender and smoking status approximating the expected characteristics of the HIV-positive population being sampled. Blood samples from Caucasian male participants were taken in Tempus tubes for storage at -80Â° until RNA. Total blood RNA was extracted using the Tempus Spin RNA Isolation Kit (Ambion; Life Technologies). Globin mRNA and genomic DNA were removed using the GlobinClear kit (Life Technologies) and the TURBO DNAfree kit (Ambion, Life Technologies), respectively. cDNA libraries were generated using the Kapa mRNA Hyperprep kit (Roche).