title: GSM8656237: SupT1 infected, Nanopore, 8hpi, rep1; Homo sapiens; Human immunodeficiency virus 1; OTHER
library_construction_protocol: Nanopore: To analyze the HIV transcriptome in infected SupT1 cells at 8, 16 and 24 hpi 30 μg of Trizol extracted RNA was treated with 3 μL of Turbo DNase in 150 μL total volume for 30 min at 37 °C. RNA was purified with Macherey-Nagel PCR Clean-up kit using NTC buffer according to manufacturer's instructions. DMS-Seq: For in vivo DMS modification of HIV-1 gRNA, 40 mL of SupT1 cells were infected with VSV pseudotyped HIV-1 (ΔEnv). 24 h post infection, 5 mL of cells were probed with 85 mM of dimethyl sulfate (DMS) for 6 min at 37°C, placed immediately on ice and the reaction was quenched by addition of 100 mM of β-mercaptoethanol. Cells were pelleted by centrifugation for 5 min at 400 g, 4°C and washed with PBS. The cell pellet was directly resuspended in 1 mL of TRI reagent (Sigma-Aldrich) and total RNA was extracted according to the manufacturer's instructions. Ten μg of total cellular RNA were treated with 4 U of TURBO DNase (ThermoFisher Scientific) for 30 min at 37°C. The RNA was purified using NucleoSpin Gel and PCR Clean- Up (Macherey-Nagel, NTC buffer), according to the manufacturer´s instructions. Nanopore: 1 ug of RNA was reverse transcribed with SuperScript IV (Invitrogen) using primer GTACAGGCAAAAAGCAGCTGCT according to manufacturer's instructions in a 10 μL reaction. The cDNA was purified with Macharey-Nagel PCR Clean-up kit using NTC buffer and eluted in 20 μL H2O. RNA was digested in a 10 μL reaction containing 3 μL cDNA product with 0.5 μL RNase A (NEB) and 0.5 μL RNase T1 (NEB) and incubation at 37 °C for 30 min, followed by column purification as described above. Next, PCR amplification of 20 μL of the RNase-digested, purified cDNA in a 100 μL reaction with Primestar GXL (Takara) using primers GGTCTCTCTGGTTAGACCAGATCTGAG and GTACAGGCAAAAAGCAGCTGCT was performed with 12 cycles of denaturation at 98 °C for 10 sec, annealing at 55 °C for 15 sec and extension at 68 °C for 2 min. PCR products were purified with a 1:1 ratio of Mag-Bind TotalPure NGS beads (Omega Bio-Tek) according to the manufacturer's recommendations and eluted in 14 μL H2O. Next, 80 ng PCR product in 5 μ was end repaired by addition of 0.7 μL NEBNext End Repair buffer (NEB) and 0.3 μL NEBNext End Repair Enzyme Mix (NEB), mixed thoroughly by pipetting and incubated for 5 min at 25 °C followed by 5 min at 65 °C. Nanopore native ligation barcoding barcodes (ONT SQK-NBD114.96) were ligated by transfer of 3.5 μL of end-repaired product into a new well, followed by addition of 1.5 μL of barcode and 5 μL of Blunt/TA ligation master mix (NEB). After incubation at 25 °C for 20 min, 1 μL of EDTA (SQK802 NBD114.96) was added, samples were pooled and purified with 0.4 volumes of Ampure XP beads (SQK-NBD114.96). Barcoded DNA was eluted in 31 μL of H2O. To ligate the motor protein 30 μL of the barcoded DNA was mixed with 10 μ of 5x Quick Ligation Buffer (NEB), 5 μL of NA and 5 μL of high concentration T4 DNA ligase (NEB) was added. After incubation for 20 min at 25 °C, 0.4 volumes of Ampure XP beads (SQK-NBD114.96) were added, beads were washed twice with 125 μL Short Fragment Buffer (SFB, SQK-NBD114.96) and eluted in 14 μL of Elution Buffer (EB, SQK-NBD114.96). DNA concentration was quantified with AccuClear Ultra High Sensitivity dsDNA Quantitation Solution (Biotium) and 10 fmol was prepared for sequencing on a R10.4.1 Flongle flow cell (FLO-FLG114) by addition of 15 μL sequencing buffer (SB) and 10 μL loading beads (LIB). Data was acquired on a MinION Mk1b with MinKNOW version 23.04.3. DMS-Seq: Reverse transcription was performed on 1 μg of purified RNA mixed with 0.5 mM dNTPs, 0.25 μM of RT primer (5´- CTT GTC TAT CGG CTC CTG CTT C - 3´) in a final volume of 12 μL. The RNA was denatured at 65°C for 5 min and placed on ice. The reverse transcription reaction was performed by addition of 1 mM of MnCl2, 1x MarathonRT buffer (Tris-HCl 50 mM, pH 8.3; KCl 200 mM; Glycerol 20%), 0.2 μL RNasin (Promega) and 40 U of MarathonRT 93 in 25 μL final volume. The reactions were incubated for 8 h at 42°C. Newly synthesized cDNAs were diluted 8 times and PCR amplified: 10 μL of diluted cDNA were mixed with 0.2 mM dNTPs, 0.25 μM of both Forward (5´ - TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CTA CCA TAA TGA TAC AGA AAG GC - 3´) and Reverse (5´ - GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGT CTC TTC CCC AAA CCT GAA G - 3´) primers, 10 μL of Q5 Buffer 5x, and 1 U Q5 polymerase (NEB) in 50 μL final volume. The initial denaturation was performed at 98 °C for 2 min, followed by 25 cycles of 10 sec at 98°C, 20 sec at 55°C and 30 sec 721 at 72°C before a final extension at 72°C for 5 min. Amplicon quality was controlled on an agarose gel 1%. DNA amplicons were purified by solid-phase reversible immobilization (SPRI) using Mag-Bind TotalPure NGS beads (Omega Biotek) following the manufacturer´s instruction, with a DNA to beads ratio of 1:0.8. Nextera indexes were added by PCR, 80 ng of purified DNA were mixed with 1x Q5 Buffer, 0.2 mM dNTPs, 0.02 U of Q5 polymerase and 2.5 μL of Nextera Index (Illumina) in a 14 μL reaction. The initial denaturation was performed at 98 °C for 2 min, followed by 5 cycles of 30 sec at 98°C, 30 sec at 62°C and 1 min at 72°C before a final extension at 72°C for 5 min. Quality of libraries was assessed by agarose gel electrophoresis. Equal amounts of each sample were pooled together followed by a final SPRI purification (DNA to beads ratio of 1:0.8). Paired-end PE150 sequencing was carried out on an Illumina Novaseq instrument (Novogene).