title: GSM9361173: Primary T cells, mock infected, donor 1, 96 h, replicate 1; Homo sapiens; OTHER
library_construction_protocol: Total RNA was isolated using TRIzol reagent according to the manufacturer's protocol. Following phase separation with chloroform, RNA was precipitated with isopropanol, washed with 75% ethanol, and dissolved in RNase-free water. First, 2 µg total RNA was mixed with 1 µl of 100 µM 3' Illumina_4N_21T_VN primer (5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNTTTTTTTTTTTTTTTTTTTTTVN-3'), 2 µl of 5 mM AzNTP (Baseclick, #BCT–25 to −28)/dNTP mixture, at a ratio of 1 to 5, and the total volume was raised to 13 µL by the addition of H2O. The mixture was heated at 65°C/5 min and snap-cooled on ice for 3 min. Reverse transcription was performed using SuperScript III (ThermoFisher, #18080093). In brief, 7 µl master mix containing 4 µl 5× Superscript First Strand buffer, 1 µl of 0.1 M DTT, 1 µl RNaseOUT (ThermoFisher, #10777019), and 1 µl SuperScript III were mixed with the RNA sample. Heating was performed in a thermocycler sequentially as follows: 25°C/10 min, 50°C/40 min, and 75°C/15 min, followed by RNase H (ThermoFisher, #AM2293) treatment using 1U per reaction for 37°C/30 min and then 80°C/10 min. The resulting cDNA was purified using Sera-Mag Speedbeads (Cytiva, #65152105050250). Speedbeads working solution was made by washing 1 ml bead slurry twice in 1×TE buffer and then resuspending beads in 50 ml of 1×TE buffer containing 9 g PEG-8000, 1 M NaCl, and 0.05% Tween-20. Speedbeads, 1.8× reaction volume, was mixed with cDNA followed by a 5 min incubation at room temperature. The beads were then pelleted using a magnetic bead collector, and the supernatant was discarded. Two washes with 80% ethanol were performed while the beads were pelleted on the magnet. The beads were then dried, and the cDNA was eluted by resuspending the beads in 22 µl of 50 mM HEPES pH 7.2 for 2 min at room temperature. Then, 20 µl cDNA was mixed with 11 µl Click Mix (2.5 M NaCl, 30% EtOH in H2O) and 4 µl of 5 µM UMI-click-adapter (5'-Hexynyl-NNNNNNNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3', HPLC purified). To activate the reaction, 4 µl of 50 mM vitamin C and 1 µl of Click Catalyst (4 mM CuSO4 in H2O and 20 mM THPTA [Baseclick, #BCMI-006] in H2O) were pre-mixed before being added into the cDNA solution for 60 min incubation at room temperature in the dark. Clicked cDNA was then purified in the same manner as post-RT purification but eluted in 22 µl of 10 mM Tris pH7.4. Half of the eluted cDNA was subjected to PCR amplification with indexing primers i5 (5'-AATGATACGGCGACCACCGAGATCTACAC[Index]ACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3') and i7 (5'-CAAGCAGAAGACGGCATACGAGAT[Index]GTGACTGGAGTTCAGACGTGTGCTCTTCCGAT*C-3'). Both primers have a phosphorothioate bond added at the 3' end (denoted as *) to increase stability. The PCR reaction was conducted in a 50 µl volume, including 25 µl 2× OneTaq Master Mix (NEB, #M0482), 2 µl each of 5 µM i5 and i7, and cDNA in H2O. A total of 13 amplification cycles were conducted on 2 µg input RNA. The program was run at 94°C/4 min; 53°C/30 sec; 68°C/10 min; 15× [94°C/30 sec, 53°C/30 sec; 68°C/2 min]; 68°C/5 min; hold at 4°C. The library was then purified and size-selected with Speedbeads. In brief, 45 µl beads (0.9× volume of PCR reaction) were mixed with the reaction to remove larger fragments. Supernatant was collected and mixed with 10 µl beads (0.2× volume of PCR reaction) to remove smaller fragments and primer dimers. Two washes with 100% ethanol were then performed while the beads were on the magnet. Beads were dried before being resuspended in 12 µl of 10 mM Tris pH7.4. The library was then collected and subjected to quality control on Agilent 2200 TapeStation and analysis by Illumina NovaSeq 6000 at the Genomics Research Center at the University of Rochester.