GSM8486554: BJ cells, Untreated CR4/5 amplicon; Homo sapiens; RNA-Seq
Total RNA was extracted using Trizol following the manufacturer's protocol. 10 μg of HeLa cell derived total RNA was DNAse treated in 1X TURBO DNAse buffer with 1 μL TURBO DNAse enzyme (Thermo Fisher Scientific), followed by column-purification with DNA Clean and Concentrator-5 columns (Zymo Research) following the manufacturer protocol. 5 μg of DNAse-treated RNA was mixed with 5 pmol hTR targeting primer containing a 4-nucleotide unique molecular identifier (UMI) in a volume of 11 μL, heated to 75°C for 3 min and annealed at 35°C for 15 min. Next, 4 μL of 5X M-MLV buffer (Promega), 1 μL 0.1M DTT, 1 μL RNAsin Plus (Promega), and 1 μL TGIRT III (Ingex) were added and the mixture was incubated at room temperature for 30 min. Then, 2 μL of 10mM dNTPs were added, and the reaction was mixed and incubated at 60 °C for 2.5 h. After RT, 1 μL of 5M NaOH was added directly to the cDNA and incubated at 95°C, followed by 2.5 μL of 2M HCl and purification by DNA Clean and Concentrator-5 columns (Zymo Research) using an 8:1 ratio of DNA Binding Buffer to cDNA volume. The cDNA was mixed into a 50 μL second strand synthesis reaction containing 10 μL 5X GC Phusion Buffer (Thermo Fisher Scientific), 1 μL 10 mM dNTP, 25 pmol of a second hTR targeting primer containing a 4 nucleotide UMI, and 1 μL Phusion polymerase. The second strand synthesis reaction was incubated in a thermocycler with the following program: 98°C 2 min, 60°C 2 min, 72°C 10 min. Second strand product was cleaned up with Ampure XP beads (Beckman Coulter) at a 0.8:1 bead to sample volume ratio and eluted in 20 μL nuclease-free H2O. Illumina sequencing libraries of two partially overlapping hTR regions (Pseudoknot: nucleotides 1-286 and CR4/5: nucleotides 193-451) were generated by two successive 50 μL PCR reactions. For the first PCR, 4 μL second strand product was added to a 50 μL PCR reaction containing primers for either the pseudoknot or CR4/5 amplicon and run with the following program: 10 cycles at 66°C annealing temperature, followed by 10 cycles at a 60°C annealing temperature. Amplicons were bead cleaned with a 0.8:1 ratio and went into a second PCR of 10 cycles at an annealing temperature of 65°C to complete the sequencing adapters and multiplexing barcodes. Libraries were bead cleaned with a 0.8:1 ratio and quantified by Qubit (Thermo Fisher Scientific) and Tapestation (Agilent) for quality metrics before sequencing by 150 bp paired end reads on the iSeq100 (Illumina).