GSM6415616: sRNA_MT4_HIV_rep1; Homo sapiens; RNA-Seq
MT4 cells infected and uninfected were collected by centrifugation (225× g, 5 min, at 20 °C). Cells pellets were washed with PBS and cells were lysed with RNAzol reagent (Sigma-Aldrich). Virus-containing supernatants were filtered (0.45 μm pore size cellulose-acetate filter (VWR)), and stored at −80 °C. HIV-1 titer was measured by 10-fold serial infection of TZM-bl cells (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. John C. Kappes and Dr. Xiaoyun Wu) in triplicate, calculated by Reed-Muench method and expressed as 50% tissue culture infectious dose. Small RNA fraction was purified according to the RNAzol manufacturer's protocol. The RNA concentration was determined on NanoDrop ONE (ThermoFisher Scientific) and the RNA sample quality control was performed on a 4200 TapeStation System NAD CaptureSeq: Briefly, only short fraction of RNA from MT4 cells infected by HIV-1 and MT4 cells not infected was used for the library preparation. The most critical step is the enzyme reaction catalyzed by ADP-ribosylcyclase (ADPRC, Sigma-Aldrich), which is an enzyme specific only for NAD-capped RNA but inactive on canonical RNA. Samples not treated by ADPRC were used as a negative control for non-specifically bound RNA. 100 μg of sRNA for each sample was incubated with 4-pentyn-1-ol (10%) and ADPRC (2.5 ug) in 50 mM Na-HEPES (pH 7.0) and 5 mM MgCl2 for 30 min at 37 °C. The reaction was stopped by phenol/ether extraction and RNA was ethanol precipitated. NAD-capped RNA was then biotinylated (biotin-PEG3-azide, 250 mM) via a copper-catalyzed azide-alkyne cycloaddition (CuAAC) in a freshly prepared mixture of CuSO4 (1 mM), THPTA (0.5 mM) and sodium ascorbate (2 mM) for 30 min at 25 °C, with shaking at 350 r.p.m. The biotinylated RNA was purified by phenol/ether extraction and ethanol precipitated. Mobicol Classic columns (MoBiTec, GmbH) were assembled and 50 μl of streptavidin Sepharose (GE Healthcare) was transferred to each column. After washing the beads by adding three times 200 μL of immobilization buffer (10 mM Na-HEPES, 1 M NaCl and 5 mM EDTA, pH 7.2) to each column and centrifuge at ≥16,100 g for 1 min at rt, the beads were blocked with acetylated BSA (100 μg/ml, Sigma-Aldrich) in 100 uL of immobilization buffer for 20 min at 20 °C, with shaking at 1,000 r.p.m. After three more washes, the RNA was immobilized on the beads for 1 h at 20 °C, with shaking at 1,000 r.p.m. The beads were washed five times with 200 μL of streptavidin wash buffer (50 mM Tris-HCl and 8 M urea, pH 7.4), then equilibrated by washing three times with 200 μL of 1x standard ligation buffer (50 mM Tris-HCl, 10 mM MgCl2, pH 7.4), blocked with acetylated BSA, and washed three more times with 1x standard ligation buffer as described previously. 30 μL of ligation mixture containing adenylated RNA 3′adaptor (5 μM, rApp-CNNNNNNAGATCGGAAGAGCACACGTCTG-(C3)), 1x standard ligation buffer, DMSO (15%, Sigma-Aldrich), acetylated BSA (1.5 μg), 2-mercaptoethanol (50 mM, Sigma-Aldrich), T4 RNA ligase (15 U, Thermo Fisher Scientific), and T4 RNA ligase 2, truncated K227Q(300 U, New England BioLabs) was added to the beads and the mixture was incubate at 4 °C overnight (≥16 h). The biotinylated RNA was rebound by adding 7.5 μL 5 M NaCl and incubated for 1 hour at 20 °C, with shaking at 1,000 r.p.m. After washing the beads five times with the streptavidin wash buffer, they were equilibrated by washing three times with 1× first-strand buffer (50 mM Tris-HCl, 75 mM KCl and 5 mM MgCl2, pH 8.3), blocked with acetylated BSA, and washed another three times with 1× first-strand buffer. The beads were covered with 30 μL of reverse transcription mixture containing dNTPs (0.5 mM each,company), acetylated BSA (1,5 μg), DTT (5 mM), 1x first strand buffer, reverse primer (5 uM, CAGACGTGTGCTCTTCCGAT), and Superscript III reverse transcriptase (300 U) and incubated for 1 hour at 37 °C. The rebinding procedure was repeated and samples were washed five times with streptavidin wash buffer and three times with 1x ExoI buffer (Thermo Scientific). The beads were blocked with acetylated BSA in ExoI buffer and again washed three times with 1x ExoI buffer. The free primers were digested by adding 30 μL of 1x ExoI buffer and 30 U of ExoI (Thermo Scientific) and the mixture was incubated for 30 min at 37 °C. The beads were washed five times with streptavidin wash buffer and three times with immobilization buffer. The RNA was digested by adding 100 μl of 0.15 M NaOH solution and incubating for 25 min at 55 °C. The columns were centrifuged and washed with 100 ul of H2O. The flow-through was ethanol precipitated and the cDNA pellet was dissolved in 19 μl of Terminal deoxynucleotidyl transferase (TdT) tailing mixture containing 1x TdT buffer (Thermo Scientific) and CTP (1.25 mM). After adding 20 U of TdT, the mixture was incubated in thermocycler for 30 min at 37 °C and after the enzyme was thermally denatured by heating to 70 °C for 10 min. Then, the second adaptor was ligated on the cDNA by adding 60 μl of reaction mixture containing 1x standard ligation buffer, cDNA anchor (5 μM each strand, sense: p-CAGATCGGAAGAGCGTCGTGT-(C3), antisense: ACACGACGCTCTTCCGATCTGGG), ATP (10 μM), and T4 DNA ligase (120 Weiss U, Thermo Scientific) and incubating for 16 h at 4 °C.PCR amplification was performed with barcoded PCR primers (Table) in 25 cycles inTaq DNA Pol reaction buffer 1 × (Thermo Scientific), with 1 μM of each barcoded primer, 0.2 mM dNTPs (each) and 2.5 U of DreamTaq DNA Polymerase (Thermo Scientific) in 50 μL of total reaction mixture. Initial denaturation was performed at 95 °C for 3 min, following by annealing for 30 s at 68 °C, elongation for 30 s at 72 °C and denaturation for 30 s at 95 °C. Final extension was performed at 72 °C for 5 min. PCR reaction mixture was loaded on 2.5% agarose gel (140 V for 2 h). Fractions between 100–400 nt were cut and DNA was extracted from the gel by Monarch DNA Gel extraction Kit  (NEB). Multiplexed samples were submitted to the IMG Genomics and Bioinformatics facility for library synthesis using the NextSeq® 500/550 High Output Kit v2, 75 cycles (Illumina) and sequencing on a NextSeq 500/550, Illumina. Short RNA-seq: The library synthesis was done using the NEBNext®Small RNA Library Prep Set for Illumina with NEBNext Multiplex Oligos for Illumina (Index Primers Set 1-3) and sequenced on a NovaSeq 6000 System (SE 50 bp, 350-400 milions of reads, DS-270). Total RNA-seq: KAPA RNA HyperPrep Kit with RiboErase was used to prepare the libraries and the sequencing was performed using the NextSeq® 500/550 High Output Kit v2, 75 cycles (Illumina) and on a NextSeq 500/550, Illumina.