GSM4838746: T1; Schizosaccharomyces pombe; RNA-Seq
Total RNAs were isolated from mid-log cells using GeneJET RNA Purification Kit (Thermo Fisher Scientific) following the manufacturer's instructions for each sample. The purity and quantity of RNAs were measured using a Nanodrop 2000 spectrophotometer (Nanodrop Technologies, USA). Total RNAs were then used in further analysis. Three replicates were used for each sample and replicates were then pooled for sequencing according to their concentrations. After the total RNA extraction and DNase I treatment, magnetic beads with Oligo (dT) are used to isolate mRNA(for eukaryotes). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters. After agarose gel electrophoresis, the suitable fragments are selected for the PCR amplification as templates. The library could be sequenced using Illumina HiSeq.