title: TSS-seq of S. cerevisiae: AYR library in E1103G
design: Total RNA was extracted by a phenol-chloroform method (Schmitt, Brown, & Trumpower, 1990), followed by RNA purification (RNeasy Mini kit, QIAGEN, 74104) with on-column DNase digestion (RNase-Free DNase Set, QIAGEN, 79254) to remove DNA. TSS-seq was done using procedures described in (Vvedenskaya, Goldman, & Nickels, 2015). To prepare RNAs for the cDNA library construction, samples were sequentially treated with Terminator 5-Phosphate-Dependent Exonuclease, calf-intestine alkaline phosphatase and Cap-Clip Acid Pyrophosphatase to remove 5' monophosphate RNA and convert 5' triphosphate or capped RNAs to 5' monophosphate RNAs. Next, RNA prepared with enzymatic treatments was ligated to the 5'-adapter (s1206-N15, 5'-GUUCAGAGUUCUACAGUCCGACGAUCNNNNNNNNNNNNNNN-3') that contains Illumina adapter sequence and a 15 nt randomized 3'-end to reduce ligation bias and serve as a Unique Molecular Identifier (UMI). Next, cDNA was constructed by reverse transcription using RT primer CKO2191-s128A (5'-CCTTGGCACCCGAGAATTCCAAGTGAATAATTCTTCACCTTTA-3') followed by PCR amplification using Illumina PCR primers (RP1 and RPI3-30). Final DNA was gel size selected for 180-250 bp lengths and sequenced by Illumina NextSeq 500 (150 SE) or NovaSeq 6000 (200 SE) using custom primer s1115 (5'-CTACACGTTCAGAGTTCTACAGTCCGACGATC-3') to avoid potentially confounding effects of misannealing of the default pooled Illumina sequencing primer to the two randomized sequence regions.