Extensive divergence of yeast stress responses through transitions between induced and constitutive activation
Equal mass of polyA+ RNA of different yeasts from the same growth conditions was pooled together and then subjected to the following treatments. First strand cDNA synthesis was performed using the Invitrogen SuperScript III RT system. Approximately 100 µg of the mixed polyA+ RNA was concentrated, when needed, by speedvac to 100 µl volume and was fragmented by heating at 95°C for 5 minutes. Subsequently, the fragmented polyA+ RNA was mixed with 2 µg of random hexamers and 10 µl of 10 mM dNTP, incubated at 65°C for 5 minutes and then chilled on ice while the reaction mix (40 µl of Invitrogen Superscript III 5x first-strand buffer, 10 µl of 0.1M DTT) was added. Actinomycin D (Sigma) was included in the reverse transcription reaction at a final concentration of 9 µg/µl to suppress the synthesis of second-strand cDNA by reverse transcriptase (RT). The mixture was incubated at 15°C for 15 minutes before the addition of 10 µl of RNaseOUT (Invitrogen) and 2000 U of Invitrogen SuperScript III RT. The reaction was incubated at 25°C for 10 minutes, 40°C for 40 minutes, 50°C for 50 minutes and 70°C for 10 minutes. Upon completion of the reaction, samples were treated with 1 µl of RNase cocktail (Ambion) and RNase H (Invitrogen) at 37°C for 30 minutes. First-strand cDNA was cleaned up twice using Performa Spin Columns (EdgeBio) with each sample loaded onto four separate columns in each round of purification. The purified cDNA was concentrated by speedvac to a volume of about 50 µl and quantified by Nanodrop.   For library preparation for sequencing using the HeliScope sequencer, 100 ng of cDNA was mixed with 5 µl of Helicos PolyA Control Oligonucleotides A, heated to 95°C for 5 minutes and quickly cooled on ice for 2 minutes. The following reaction mix (5 µl each of CoCl, 10X TdT buffer and PolyA tailing dATP (Helicos) and 2 µl of terminal transferase (NEB)) was then added before incubating at 42°C for 1 hour, 70°C for 10 minutes and 4°C until the next step. A final 3’ blocking step was performed on the polyA-tailed cDNA sample by heating at 95°C for 5 minutes, then cooling on ice for 2 minutes before the addition of 0.4 µl of biotin-ddATP (PerkinElmer) and 2 µl of Terminal transferase (NEB). The blocking reaction was carried out at 37°C for 1 hour and Terminal transferase was inactivated at 70°C for 10 minutes. The resultant library was kept at 4°C in dark and was quantified and sequenced using the HeliScope sequencer at the Molecular Biology Core Facility of Dana Farber Cancer Institute.