title: GSM6675680: Trehalose_t-5_rep2; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Total RNA was isolated by the standard phenol chloroform method and DNA was removed by DNase I treatment, as described in Zhang & Pelechano, 2021 (PMID:33870233 ) :   The amount of RNA was quantified using  a Nanodrop 2000 Spectrophotometer (Thermo Scientific) and RNA integrity was checked by agarose gel electrophoresis. For HT5Pseq libraries construction, we used 6 μg of DNA-free total RNA. Samples were directly subjected to RNA ligation. The treated RNA samples were incubated with 100 μM RNA/RNA rP5_RND oligo (final 10 μM) 2h at 25°C with 10 Units of T4 RNA ligase 1 (NEB). Ligated RNA was purified with RNAClean XP (Beckman Coulter), according to the manufacturer's instructions. RNA was reverse transcribed with Superscript II (Life Technologies) and primed with Illumina PE2 compatible oligos containing random hexamers (20 μM) and oligo-dT (0.05 μM). Reverse transcription reaction was incubated for 10 min at 25°C, 50 min at 42°C and heat inactivation for 15 min at 70°C. To deplete RNA in RNA/cDNA hybrid after reverse transcription, we used sodium hydroxide (40 mM) for incubation 20 min at 65°C and then neutralized with Tris-HCl, pH =7.0 (40 mM). For DSN (Duplex-specific nuclease) based rRNA depletion, we used a mixture of probes targeted the 18S rDNA, 25S rDNA and 5.8S rDNA. The probes were designed to occupy the whole ribosomal RNA regions with consecutive 25-30nt long unmodified DNA oligos. The hybridization of probes (2 μM each) with cDNAs were incubated at 68 °C for 2 minutes before adding pre-warmed DSN buffer mix with 1 Units of DSN enzyme (Evrogen). The reaction then performed at 68 °C for 20 minutes. To inactive DSN enzyme, we added 2X DSN stop solution and incubate 10 min at 68 °C. The final PCR amplification was used 2X Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB) and final 0.1 μM of PE1.0 and corresponding multiplex PE2.0_MTX . The program followed this: 30s 98°C; 15 cycles (20s 98°C; 30s 65°C; 30s 72°C); 7min 72°C. Libraries were size selected using 0.7x-0.9x (v/v) AMpure XP beads (Beckman Coulter) to final 200-500 bp and sequenced by NextSeq 2000 using 55 sequencing cycles for Read 1.  5PSeq methods was performed as previously described (PMID: 26820793). 6µg of total RNA was used as input. In brief a RNA oligo (rP5_RND) containing an Illumina adaptor and unique molecular identifiers (UMI) was ligated to the intermediates of mRNA co-translation degradation (5'P). Ribosomal RNA was depleted using Ribo-Zero Magnetic Gold Kit (Illumina).Libraries were PCR amplified (15 cycles). Ampure beads size selected libraries with an average length of 400 nt were sent for sequencing (llumina NextSeq 2000 instrument).