GSM9455678: 11BH Jurkat HIV-GFP (RNaseH Only); Human immunodeficiency virus 1; OTHER
Total RNA was harvested via TRIZol (Invitrogen), using the manufacturer's protocol. After extraction, total RNA was divided, and approximately 15-20 μg was used for poly-A selection and RNaseH ribosomal RNA depletion. For poly-A selection, the NEBNext High Input Poly(A) mRNA Isolation Module (E3370S) was used according to the manufacturer's protocol. For the RNaseH treatment, total RNA was hybridized with DNA oligonucleotides complementary to rRNA. The solution was heated to 95°C and slow cooled to 65°C to enable oligonucleotide hybridization. RNase H (McLabs, HTRH-200) was added (200 U) and the solution incubated at 65°C for 5 min to degrade rRNA. DNA was then degraded using 150 U of Turbo DNase (Invitrogen, AM2239) at 37°C for 20 min. RNA was then purified using AMPure XP beads. A polyadenylation reactions was performed using E. coli Poly(A) Polymerase (New England Biolabs, M0276L).  The final library was loaded on a PromethION RNA flow cell (FLO-PRO004RA) and sequenced on a PromethION P2 solo using MinKNOW (v. 24.06.10) with POD5 and live base-calling on.