GSM8865281: Top1/2-AID + TIR1 0h, R2; Saccharomyces cerevisiae; OTHER
24 OD units were collected by centrifugation, and snap frozen in liquid nitrogen. Per 10 OD units, RNA was extracted with Tris-EDTA-SDS (TES) buffer (10 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.5% SDS) and Acid Phenol:Chloroform:Isoamyl alcohol (125:24:1, Ambion) at 65°C for 45 minutes. Precipitation was carried out. rDNase (Machery-Nagel) treatment was carried out, before spin column purification (Machery-Nagel). The TSS-seq protocol: total RNA was dephosphorylated with quickCIP (NEB, 1.2 U/µg RNA) 37°C, with RNasin Plus before heat inactivation. RNA was extracted using phenol/chloroform/isoamyl alcohol as before. RNA was treated with mRNA decapping enzyme (NEB, 0.7 U/µg RNA) at 37°C with RNasin Plus. RNA was extracted using phenol/chloroform/isoamyl alcohol as before. RNA and the 5' adaptor (10 µM) were denatured for 2 minutes at 70°C, then cooled on ice for 2 minutes. Ligation was performed, 16°C using and T4 RNA ligase I (30 U), with RNasin Plus. RNAClean XP (1.8X ratio, Beckman Coulter) was used to remove unligated adaptors, according to the manufacturer's instructions. The concentration of the samples was measured using Qubit RNA BR assay, and samples were multiplexed. polyA+ RNA was enriched using Dynabeads Oligo(dT)25 (Thermo Fisher). Yeast RNA was fragmented with alkaline fragmentation reagent (Ambion). RNeasy MinElute Clean-up columns, with 1.5x volume of Ethanol, were used to purify the samples. The 3' ends were fixed by CIP treatment (NEB,30 U).  Ligation was carried out by T4 Rnl2tr (NEB,200 U) for 2 hours, 25°C then 16 hours, 16°C. RNAClean XP (1.8X ratio) was used to remove unligated adaptors. RNA, RT oligo (0.5 pmol) and dNTP (10 mM each) were denatured at 65°C, then cooled on. Reverse transcription was carried out by SuperScript III (2 µl), with RNasin Plus with the following conditions: 25°C for 5 minutes, 42°C for 20 minutes, 50°C for 40 minutes, 80°C for 5 minutes. Template RNA was removed by RNase H (10 U) for 37°C, 30 minutes. AMPure XP beads (Agencourt, Beckman Coulter) were used to clean up the cDNA. 3X beads and 1.7x isopropanol added to the reverse transcription reaction, and incubated for 5 minutes. Beads were washed twice with 85% ethanol, dried, and eluted twice in nuclease-free water. cDNA pre-amplification was performed with i5_s and i7_s (300 nM each) primers and Phusion HF PCR Mastermix (Thermo Fisher). 6 PCR cycles were carried out (98°C for 10 seconds, 65°C for 30 seconds, 72°C for 30 seconds, with 3 minutes at 72°C final extension). ProNex beads (Promega) with a 1:2.95 ratio to sample were used to remove sequences less than 55 nt (including primer dimers). Final amplification was carried out with NEBNext i50 and i70 primers (500 nM each) and Phusion HF PCR Mastermix, for 8 cycles. Amplification was checked by using Novex 6% TBE (Tris/Borate/EDTA) gel and staining with SYBR green. ProNex beads (Promega) with a 1:2.3 ratio to sample were used for purification. Library concentration was measured using Qubit High Sensitivity D1000. Libraries were sequenced with Illumina NovaSeq 6000.