GSM6912107: Yeast_HPRT1R_YAC_CAGE_rep2; Saccharomyces cerevisiae; RNA-Seq
Yeast were pelleted at 3,000 rcf for 5 minutes, washed once with water, pelleted again at 3,000 rcf for 5 minutes, and cell pellets were frozen at -80oC. Frozen pellets were resuspended in 200 μL lysis buffer (50 mM Tris-HCl pH 8, 100 mM NaCl) and lysed by disruption with an equal volume of acid washed glass beads, vortexing 10 x 15 seconds. 300 μL lysis buffer was added and samples were mixed by inversion followed by a short centrifugation to collect all liquid in the tube. 450 μL of supernatant was mixed with an equal volume of phenol:chloroform:isoamyl alcohol, vortexed for 1 minute, and centrifuged at max speed for 5 minutes. 350 μL of the aqueous layer was then mixed with an equal volume of phenol:chloroform:isoamyl alcohol, vortexed for 1 minute, and centrifuged at max speed for 5 minutes. RNA was precipitated from 300 μL of the aqueous phase by adding 30 μL of 3 M NaOAc and 800 μL of cold 99.5% ethanol, briefly vortexing, and centrifuging at max speed for 10 minutes. The pellet was rinsed with 70% ethanol and dried at room temperature before dissolving in 100 μL of RNase-free DNase set (Qiagen 79254) and incubating at room temperature for 10 minutes to remove DNA. RNA was purified using the RNeasy Plus Mini kit (Qiagen ​​74136) and eluted in 30 μL RNase-free water. CAGE libraries were prepared as previously described (Murata, M., et al., Methods Mol Biol, 2014), starting with 5 μg RNA, with the following modifications. SuperScript™ IV Reverse Transcriptase (Invitrogen 18090010) was used for the reverse transcription step. AMPure XP beads (Beckman Coulter A63881) were used for all bead cleanup steps. We also used custom-made linker and primer oligos so that linkers are universal to all samples and primers contain sample-specific barcodes. Libraries were amplified using universal forward and reverse primers with 20 cycles of PCR