GSM2574053: RNA_S1; Streptomyces tsukubensis; RNA-Seq
For RNA-seq experiments, cells were harvested in early-exponential phase (15 h), transition phase (18.5 h), late-exponential phase (28 h), and stationary phase (48 h). Total RNA was isolated from obtained cell lysate by phenol-chloroform extraction and ethanol precipitation. Isolated total RNA was incubated at 37oC for 1 h with DNase I (New England Biolabs) to remove genomic DNA. Total RNA was further purified by phenol-chloroform extraction and ethanol precipitation. Quality of the isolated total RNA was checked by visualization using 2% agarose gel electrophoresis. Ribosomal RNA was specifically removed using Ribo-ZeroTM kit (Epicentre). Strand-specific RNA sequencing libraries were constructed using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina Inc) according to manufacturer’s instructions. Briefly, mRNA samples were chemically fragmented into small pieces and reverse transcribed into cDNA using reverse transcriptase and random primer. The cDNA samples’ ends were repaired and A-tailed to both ends of the cDNA. Illumina adaptors were ligated to A-tailed cDNA and the ligated products were PCR-amplified.