GSM8626469: CoSTseq, dst1∆, dms treated, biol rep 3; Saccharomyces cerevisiae; OTHER
Cells were pelleted at 3,500×g and washed with stop buffer without isoamyl alcohol, then resuspended in 600 µl lysis buffer (45 mM NaOAc, 6 mM EDTA, pH 5.5) with 40 µl 20% SDS added after resuspension. The mixture was then incubated at 65 ºC for 30 s with vigorous shaking, added to 650 µl hot (65 ºC) acid-phenol:chloroform:IAA 125:24:1, pH 4.5 and incubated for 2 min at 65 ºC with shaking. Samples were placed on ice for 5 min, 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 80 µg 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 (2 M NaCl, 50 mM Tris⋅HCl pH 7.4, 0.5% Triton X-100), once with binding buffer (300 mM NaCl, 10 mM Tris⋅HCl pH 7.4, 0.1% Triton X-100), and once with low salt buffer (5 mM Tris⋅HCl pH 7.4, 0.1% Triton X-100); 500 µl each. Nascent RNA was extracted from beads twice using Trizol and chloroform and resuspended in 3.75 µl H2O. Reverse transcription was performed in a 10 µl volume with an optimized KCl based buffer made of 75 mM KCl, 3 mM MgCl2, 50 mM Tris⋅HCl pH 8.3 and 0.5 µl RNase inhibitor using the GsI2c-MBP fusion protein (commercially TGIRT-III), which was purified as described previously. RT reactions were pre-incubated with 100 nM annealed DNA/RNA heteroduplex adapter with a single G overhang to capture the biotin-C at the 3'-end (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTG, rArArGrArUrCrGrGrArArGrArGrCrArCrArCrGrUrCrUrGrArArCrUrCrCrArGrUrCrArC/3SpC3/) for 30 min at room temperature. Then 1.25 µM dNTP were added and the reaction was incubated at 25 °C for 10 min, 42 °C for 10 min, 50 °C for 10 min, 55 °C for 10 min, 60 °C for 30 min, 65 °C for 20 min, and 75 °C for 15 min. 1 µl of RNase H (NEB) was added and the sample incubated at 37 ºC for 30 min, following clean-up using MinElute PCR Purification kit (Qiagen). The 5'-end adapter (/5Phos/NNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT/3SpC3/) was first adenylated using Mth RNA Ligase (NEB), purified using Oligo Clean & Concentrator (Zymo Research) and then ligated to cDNA using Thermostable 5' App DNA/RNA Ligase (NEB) according to the manufacturer's instructions. Adapter ligated cDNA was again cleaned using spin column purification. PCR was carried out with KAPA HiFi HotStart 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.