GSM8223146: ntg_TNF1; Homo sapiens; RNA-Seq
Cells were washed 1x with 3 mL PBS then RNA harvested in 1 mL Trizol First, 5 µg total RNA was subjected to poly(A) mRNA isolation according to the manufacturer's protocols (NEB, #E7490). Then, 10 µL of the eluted mRNA was mixed with 1 µL 100 µM Genomic Adaptor_6N primer (5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNN-3') and 2 µL of 5 mM AzNTP (Baseclick, #BCT-25~ -28)/dNTP mixture in a 1:35 ratio.  The mixture was then heated at 65°C/5min and snap-cooled on ice for 3min. Then, the reverse transcription was carried out using SuperScript III (ThermoFisher, #18080093). In brief, 7 µL master mix containing 4 µL 5X Superscript First Strand Buffer, 1 µL 0.1 M DTT, 1 µL RNaseOUT (ThermoFisher, #10777019), and 1 µL Superscript III was mixed with the RNA sample. Heating was performed as follows: 25°C/10 min, 50°C/40 min, and 75°C/15 min sequentially in thermocycler followed by RNase H (ThermoFisher, #AM2293) treatment using 1 U per reaction for 37°C/30 min and then 80°C/10 min. The cDNA was then purified using Sera-Mag Speedbeads (Cytiva, #65152105050250). Speedbeads working solution was made by washing 1 mL beads slurry twice in 1XTE buffer and then resuspending in 50 mL of 1XTE buffer containing 9 g PEG-8000, 1 M NaCl, and 0.05% Tween-20. Speedbeads, 1.8x 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 50 mM HEPES pH 7.2 for 2 min at room temperature. Then, 20 µL of the cDNA was mixed with 11 µL Click Mix 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 were pre-mixed before being added into the cDNA solution for 60min room temperature incubation in the dark. Clicked cDNA was then purified in the same manner as post-RT purification except eluted in 22 µL of 10 mM Tris pH 7.4. Then, half of 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 carried out in the volume of 50 µL including 25 µL 2X OneTaq Master Mix (NEB, #M0482), 2 µL of 5 µM i5 and i7 each, and cDNA in H2O with a total of 15 cycles of amplification given the 5 µg input RNA. The program was run at 94°C/4 min; 53°C/30 sec; 68°C/10 min; 15X [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 (0.9X volume of PCR reaction) were mixed with the reaction to remove larger fragments. The supernatant was collected and mixed with 10 µL beads (0.2X 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 pH 7.4. The library was then collected and subjected to quality control on Agilent 2200 TapeStation Poly(A)-ClickSeq (PAC-seq) library preparation