GSM3597831: Yeast_total_RNA_rep2; Saccharomyces cerevisiae BY4741; RNA-Seq
Total RNA was extracted from 10 mL of yeast cells (OD600 = 0.6) using a standard hot acidic phenol method. Total RNA samples were treated with RNase-free DNase I (1 U/µg) to remove residual genomic DNA. poly(A) RNA isolation was performed in two sequential steps. In the first step oligo-dT magnetic beads were used to selectively bind poly-adenylated RNAs; these beads hybridize to the poly(A) sequence terminating the 3' end of eukaryotic mRNAs. In the second step, we used a commercial rRNA depletion kit (RiboZero Gold) to remove the residual 5S, 5.8S, 18S and 28S rRNAs from our samples. After quality and quantity measurements by using 2100 BioAnalyzer, 50 ng of total RNA and poly(A) RNA were fragmented using fragmentation buffer with the TruSeq mRNA Sample Preparation Kit according to manufacturer's instructions. Briefly, fragmented RNAs fragments were used for the first-strand cDNA synthesis using random hexamer-primer, and then the second strand was synthesized by using DNA polymerase I and RNase H. The double strand cDNAs were purified with AMPure XP beads and eluted with resuspension buffer followed by end-repair and 3'end adenine nucleotide addition. Finally, indexing adaptors were ligated to the fragments and cDNA fragments were enriched by 14-cycles of PCR amplification. Single-end sequencing was performed for the two cDNA libraries (two biological replicates) for each sample (poly(A) RNA and total RNA) using the Illumina HiSeq 4000 sequencing platform.  All sequence data are SE 50 bp.  Image processing, base calling, and quality value calculation were performed by the Illumina data processing pipeline. Raw reads were saved in FASTQ format.
GEO Accession
GSM3597831