title: GSM8036962: CoSTseq, wildtype, untreated, biol rep 2; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Cells were pelleted at 3,500×g and washed with stop buffer without isoamyl alcohol, then resuspended in 600µl lysis buffer (45mM NaOAc, 6mM EDTA, pH 5.5) with 40µl 20% SDS added after resuspension. The mixture was then incubated at 65ºC for 30s with vigorous shaking, added to 650µl hot (65ºC) acid-phenol:chloroform:IAA 125:24:1, pH 4.5 and incubated for 2min at 65ºC with shaking. Samples were placed on ice for 5min, then spun at 20,000×g at room temperature. The aqueous layer was subsequently again extracted with acid-phenol, then with chloroform and RNA was precipitated with isopropanol. CoSTseq: Approximately 80µg of input RNA were incubated with Dynabeads MyOne Streptavidin C1 (Invitrogen) according to the manufacturer's recommendations. The supernatant of the pulldown was retained for mRNA isolation. Beads were washed twice with high salt buffer (2M NaCl, 50mM Tris⋅HCl pH 7.4, 0.5% Triton X-100), once with binding buffer (300mM NaCl, 10mM Tris⋅HCl pH 7.4, 0.1% Triton X-100), and once with low salt buffer (5mM Tris⋅HCl pH 7.4, 0.1% Triton X-100); 500µl each. Nascent RNA was extracted from beads twice using Trizol and chloroform and resuspended in 3.75µl H2O. Reverse transcription was performed in a 10µl volume with an optimized KCl based buffer made of 75mM KCl, 3mM MgCl2, 50mM Tris⋅HCl pH 8.3 and 0.5µl RNase inhibitor using the GsI2c-MBP fusion protein (commercially TGIRT-III), which was purified as described previously. RT reactions were pre-incubated with 100nM annealed DNA/RNA heteroduplex adapter with a single G overhang to capture the biotin-C at the 3'-end (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTG, rArArGrArUrCrGrGrArArGrArGrCrArCrArCrGrUrCrUrGrArArCrUrCrCrArGrUrCrArC/3SpC3/) for 30min at room temperature. Then 1.25µM dNTP were added and the reaction was incubated at 25°C for 10min, 42°C for 10min, 50°C for 10min, 55°C for 10min, 60°C for 30min, 65°C for 20min, and 75°C for 15min. 1µl of RNase H (NEB) was added and the sample incubated at 37ºC for 30min, following clean-up using MinElute PCR Purification kit (Qiagen). The 5'-end adapter (/5Phos/NNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT/3SpC3/) was first adenylated using MthRNALigase (NEB), purified using Oligo Clean & Concentrator (Zymo Research) and then ligated to cDNA using Thermostable5'AppDNA/RNALigase (NEB) according to the manufacturer's instructions. Adapter ligated cDNA was again cleaned using spin column purification. PCR was carried out with KAPAHiFiHotStart polymerase (Roche) using Illumina TruSeq index primers (i5: AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACG, i7: CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTG) for 16-20 cycles. Final libraries were size selected in the 200-1,000 bp range by cutting from a polyacrylamide gel. DMS-MaPseq: RNA was precipitated from the supernatant of the biotin pulldown using ethanol. 50 µg were used as input for the Dynabeads Oligo(dT)25 mRNA purification kit (Invitrogen) and mRNA was purified according to the manufacturer's instructions. RNA was then fragmented using 10 mM ZnCl2 at 94 ºC for 55 s and precipitated using isopropanol. End repair using T4 PNK was performed according to the manufacturer's instructions. Fragmented mRNA was then size selected by differential silica binding with RNA Clean & Concentrator-5 spin columns (Zymo Research) using 33% ethanol. RT was carried out as described above, except the DNA strand contained a random overhang (N) instead of a G. Adapter ligation, PCR and sequencing were performed as described above. Mature rRNA DMS-MaPseq was carried out by fragmentation and library preparation without any prior enrichment steps.