GSM3389535: JGY2000 grown in YPD NAI-treated SHREADS Biological Replicate B; Saccharomyces cerevisiae; RNA-Seq
Hot acidic phenol extraction of total RNA, followed by purification on Qiagen Rneasy columns with Dnase I treatment DREADS/SHREADS library protocol: 25 µg mRNA was purified from total RNA samples with oligo dT(25) magnetic beads (NEB), and was then bound to beads pre-coated with a U45T5 biotinylated mRNA capture oligonucleotide1.  Immobilized mRNA tails were trimmed with RNase H to generate shortened poly(A) sequences averaging ~ 5 A's as described1.  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 B1.  cDNAs were ligated with T4 DNA Ligase to splint adapter C, and the single-stranded DNA products (110 – 500 nt) were purified on 6% polyacrylamide-urea gels.  DNA fragments were amplified by 18 cycles of PCR with barcoded oligonucleotides as described1.  Amplified libraries were purified with SizeSelector-I beads (Aline Biosciences) as described1. Adapter A: 5′-/5rApp/NNNNGATCGTCGGACTGTAGAACTCTGAAC/3ddC/-3′  Primer B: 5′ -GTTCAGAGTTCTACAGTCCGACGATC-3′  Splint Adapter: This is a double-stranded adapter where Z is the double-stranded equivalent of N.                                      5' – ZZZZTGGAATTCTCGGGTGCCAAGG – 3'                     3' – NNNNNNZZZZACCTTAAGAGCCCACGGTTCC – 5'  1. Jin, Y. et al. Mapping 3' mRNA isoforms on a genomic scale. Curr. Protoc. Mol. Biol. 110, 4.23.21-24.23.17 (2015)