title: GSM8824237: WT_4TU_9min_rep1_RATE_seq; Saccharomyces cerevisiae; OTHER
library_construction_protocol: For RNA-seq, cells were spiked with Schizosaccharomyces pombe cells at an OD ratio of 14:1, followed by RNA extraction using acid(AE-buffered)-phenol chloroform and isopropanol precipitation For RATE-seq, RNA was isolated using acid(AE-buffered)-phenol chloroform extraction, then precipitated by isopropanol and labeled RNA was captured on streptavidin beads. For Ribo-seq total RNA, RNA was isolated from cell lysates using acid(AE-buffered)-phenol chloroform followed by isopropanol extraction. For Ribo-seq ribosome-protected footpfint (RPF), cell lysates were treated with 300ug/ml RNaseI, and then resolved on 10-50% sucrose density gradient at 39k rpm and 4C for 3 hours in an SW40Ti Beckmann rotor. Sucrose gradient was fractionated using BRANDEL fractionator (SYN-202). RNA was isolated from monosome fraction by gel electrophoresis (TBE) and used to prepare ribosome-protected footprint (RPF) libraries. For steady-state RNA-seq, RATE-seq, and Ribo-seq total RNA, libraries were prepared using the Illumina™ Total-stranded RNA library preparation kit. RNA samples were rRNA-depleted with the Qiagen FastSelect™, fractionated and then reverse-transcribed. Complimentary (second) strand DNA was synthesized and cDNA libraries were PCR-amplified. Libraries were quantified on Agilent TapeStation™, then on StepOne Plus™ qPCR machine. Libraries were pooled and sequenced on Illumina NextSeq 2000™. For Ribo-seq RPF, libraries were prepared according to the widely-used Ribo-seq protocol (McGlincy and Ingolia, 2017). Custom barcodes were ligated using CircLigase I™ and cDNA was generated using Protoscript II™ reverse transcriptase. rRNA was depleted by selective hybridization, and libraries PCR-amplified.