GSM7114824: JGY2000 YPGal +DMS Rep B; Saccharomyces cerevisiae; OTHER
Total RNA was isolated and purified from 15-20 ml of cells using the hot acid phenol method followed by purification using QIAGEN RNeasy with DNase I treatment per the manufacturer's instructions. For measuring levels of 3' mRNA isoforms, 3' READS was performed with 25 mg of purified total RNA with 18 cycles of amplification as described (Jin et al., 2015).  For DREADS (Moqtaderi et al, 2018), poly(A) RNA was isolated from 25 µg of total RNA via purification with magnetic oligo dT(25) beads (New England Biolabs) and was then bound to beads pre-coated with a U45T5 biotinylated mRNA capture oligonucleotide.  Bead-immobilized mRNA tails were trimmed with RNase H (New England Biolabs) to generate shortened poly(A) sequences averaging ~5 A's as described.  After ligation of 5' pre-adenylated adapter A to the trimmed mRNA 3' ends, mRNAs were reverse transcribed with Superscript III (Life Technologies) using primer B.  cDNAs were ligated with T4 DNA Ligase (NEB) to splint adapter C, and the single-stranded DNA products (110 – 500 nt) were purified on 8% polyacrylamide-urea gels. DNA fragments were amplified by 18 cycles of PCR with barcoded oligonucleotides. Amplified libraries were purified with SizeSelector-I beads (Aline Biosciences), quantified via Bioanalyzer 2100 (Agilent Technologies), pooled, and further purified by electrophoresis on 8% polyacrylamide gels. 3' READS: Jin, Y., et al., Mapping 3' mRNA isoforms on a genomic scale. Curr. Protoc. Mol. Biol., 2015. 110: p. 4.23.1-4.23.17 DREADS: Moqtaderi, Z., J.V. Geisberg, and K. Struhl, Extensive structural differences of cloesly related 3' mRNA isoforms: links to Pab1 binding and mRNA stability. Mol. Cell, 2018. 72: p. 849-861