GSM7501618: VarsSeqRNA; Saccharomyces cerevisiae; OTHER
RNA was extracted using the YeaStar RNA Kit (Zymo Research), using 5 μL of Zymolase for 5 mL starting cell culture. 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 withrSAP (NEB), and then ligated to an adenylated universal DNA cloning linker withT4 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), using a reverse transcription primer that included a 10N interval for unique molecular identifiers (UMIs). cDNA was amplified using targeted PCR primers for each sub-library, first adding i5/i7 indices along with Illumina adapters in a short indexing PCR. We then carried out an additional PCR reaction with P5/P7 primers to generate the final library and size-selected the library using RNACleanXP beads (Beckman Coulter).