NextSeq 500 paired end sequencing; Molecular reprogramming of primary human resting memory CD4+ T cells by HIV-1 Vpr
Molecular reprogramming of primary human resting memory CD4+ T cells by HIV-1 Vpr
To infect human primary resting CD4+ T cells with HIV-1 (pNL4.3), cells were CellTrace FarRed labeled and co-cultured with eFluo450-labelled primary CD4+ T cells either infected with HIV-1 WT, HIV-1 dVpr (pNL4.3 ΔVpr) or mock (sham) treated in the presence or absence of IL7 (20 ng/mL). After 72h of co-culture, resting target CD45RA-/CD45RO+ memory T cells were recovered by flow cytometry sorting and immediately lysed in RLT buffer. Samples were stored at -80C until extraction. Total RNA was extracted from flow cytometry sorted target memory CD4+ T cells using the RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions, including the on-column DNAse treatment step. For preparation of RNA-Sequencing libraries, RNA concentration was measured using the Qubit RNA High Sensitivity kit (Life Technologies) and quality checked on the 4200 Tapestation using either the High Sensitivity or standard RNA ScreenTape assay (Agilent Technologies), depending on the measured RNA concentrations. PolyA-tailed mRNA was separated for sequencing during library preparation. Libraries were prepared using KAPA's mRNA HyperPrep kit (Roche Diagnostics) according to the manufacturer's instructions using an input of up to 200ng and a fragmentation incubation time of 8 minutes at 94°C.