GSM6402546: DMS_no_pladB; Saccharomyces cerevisiae; RNA-Seq
RNA was extracted using the YeaStar RNA Kit (Zymo Research), using 7.5 μL of Zymolase for every 2.5 mL starting cell culture and shortening the Zymolase incubation to 15 minutes at 30 oC to reduce RNA fragmentation. We first depleted the extracted RNA of rRNA using RNase H to deplete rRNA with complementary oligos. We used an RNA Clean and Concentrator-5 (Zymo) column with the size-selection protocol to exclude RNA below a size cutoff of 200 nucleotides. RNA was fragmented with Ambion fragmentation reagents, end-repaired with rSAP (NEB), and then ligated to an adenylated universal DNA cloning linker with T4 RNA ligase 2 truncated KQ (NEB). Excess DNA linker was degraded with 5' Deadenylase (NEB) and RecJf (NEB). We reverse transcribed RNA with TGIRT enzyme (InGex), and we size-selected cDNA (200 - 400 nucleotide size range) using a denaturing PAGE gel extraction to remove excess RT primer. cDNA was circularized with CircLigase ssDNA Ligase (Lucigen), and index sequences and Illumina sequencing adapters were added via PCR to generate the final library. 