title: GSM9160411: RT-Stop Profiling, RNA oligo library, 7-deaza-GTP, KCl, rep1; synthetic construct; OTHER
library_construction_protocol: 1 µg RNA of the oligo library was used as a template for the reverse transcription with SuperScript III (Invitrogen). First, the RNA was mixed with 0.5 pmol of the RT primer (5'-GGATCCTGAACCGCT-3') and 0.5 mM dNTPs in reverse transcription buffer (20 mM Tris-Cl pH 7.5, 150 mM KCl or 150 mM NaCl, 3 mM MgCl2) in a total reaction volume of 19.5 µL. The mixture was incubated at 80°C for 2 min, followed by an incubation at 25°C for 2 min. Then, 100 U of SuperScript III enzyme was added and the reaction was incubated for 10 min at 42°C. To hydrolyse the template RNA, 1.65 µL of 1 M NaOH solution was added, followed by an incubation at 98°C for 20 min. Addition of 20 µL 1M HEPES-NaOH pH 7.3 stopped the hydrolysis reaction. Then, the cDNA was isolated using a MyONE Silane bead clean-up following the iCLIP2 protocol (PMID: 31610236). The library preparation was performed using the iCLIP2 protocol continuing with second adapter ligation to the 5' end of the extracted cDNA (PMID: 31610236). The second adaptor was ligated to the 5' end of the cDNAs on the beads. Then, the cDNA was purified via MyONE Silane beads and the library was amplified using a 6-cycle PCR and afterwards two times size-selected with ProNex beads. The samples were quality checked and profiled on a High Sensitivity D1000 ScreenTape using TapeStation 2200 (Agilent). After quantification with the Qubit dsDNA HS Assay Kit (Life technologies) the samples were pooled in an equimolar ratio. Sequencing was performed on a NextSeq 500 Mid Output Flowcell using paired-end sequencing with 150 cycles (Read 1: 141nt, Read 2: 25nt).