3'-RACE of Saccharomyces cerevisiae aW303/I0 15S rRNA
Cells were grown to OD600 of approximately 0.3, the total RNA was isolated using one-step hot formamide extraction protocol as described (Shedlovskiy et al., 2017) and further purified with RNAzol RT reagent. Prior RT-PCR, the isolated RNA was dephosphorylated with T4 PNK and purified on SPRI beads. To prepare cDNA libraries for next generation sequencing, 3-terminated DNA adapter was first 5'-adenylated with Mth RNA ligase. The enzyme was removed with Proteinase K, and the adapter was purified using Zymo Oligo Clean & Concentrator kit (Zymo Research). The 3 adapter was ligated to purified RNA using T4 RNA ligase 2, truncated KQ (NEB), the cDNA was generated using RT primer complementary to the adapter and SuperScript III reverse transcriptase (Thermo Fisher Scientific), purified with SPRI beads, and used in amplification reaction (21-24 cycles) with DreamTaq 2 PCR Master Mix (Thermo Fisher Scientific) using gene-specifc and adaptor-specific oligonucleotides containing Illumina-specific sequences as recommended by Amplicon-EZ protocol (Azentha/Genewiz). The amplification products were cleaned with SPRI beads and submitted for Amplicon-EZ next-generation sequencing service (Azentha/Genewiz). The detailed protocol is published elsewhere (Anikin at al., 2024).