title: GSM1152563: Wild-type_1_2; Schizosaccharomyces pombe; RNA-Seq
library_construction_protocol: Total RNA was prepared from cells by extraction with hot acidic phenol as described previously (Collart and Oliviero 2001 Curr Protoc Mol Biol). Replicates were grown together and processed separately for all following steps. The Vanderbilt Technologies for Advanced Genomics Core Facility (VANTAGE, Nashville, TN) used the Illumina TruSeq Stranded mRNA Sample Preparation Kit to convert the mRNA in 100 ng of total RNA into a library of template molecules suitable for subsequent cluster generation and sequencing on the Illumina HiSeq 2500.  The input total RNA was quality-checked by running an aliquot on the Agilent Bioanalyzer to confirm integrity. The Qubit RNA fluorometry assay was used to measure concentration.  The input to library prep was 50 μl of 100 ng of DNase-treated total RNA (2 ng/μl). The total RNA underwent enrichment of the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads.  Following purification, the eluted poly(A) RNA was cleaved into small fragments of 120-210 bp using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using SuperScript II reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The cDNA fragments were then put through an end repair process, the addition of a single 'A' base, and ligation to the Illumina multiplexing adapters. The products were then purified and enriched with PCR to create the final cDNA sequencing library. The cDNA library underwent quality control by running on the Agilent Bioanalyzer HS DNA assay to confirm the final library size and on the Agilent Mx3005P qPCR machine using the KAPA Illumina library quantification kit to determine concentration. A 2 nM stock was created and samples pooled by molarity for multiplexing.  From the pool 12 pM was loaded into each well for the flow cell on the Illumina cBot for cluster generation. The flow cell was then loaded onto the Illumina HiSeq 2500 utilizing v3 chemistry and HTA 1.8. The raw sequencing reads in BCL format were processed through CASAVA-1.8.2 for FASTQ conversion and demultiplexing. The RTA chastity filter was used and only the PF (passfilter) reads were retained for further analysis.