RNA-Seq of perchlorate-reducing enrichment cultures
RNA library for metatranscriptome-seq was prepared as rRNA depletion andstranded method. Briefly, the ribosomal RNA was depleted from total RNA using therRNA Removal Kit following manufacturers instruction. RNA was then fragmentedinto 250~300 bp fragments and reverse-transcribed into cDNA subsequently.Remaining overhangs of double-strand cDNA were converted into blunt ends viaexonuclease/ polymerase activities. After adenylation of 3 ends of DNA fragments,sequencing adaptors were ligated to the cDNA. In order to select cDNA fragments ofpreferentially 250~300 bp in length, the library fragments were purified with AMPure XP system. Amplification of cDNA was performed using PCR. After libraryconstruction, the concentration of library was measured by the Qubit fluorometer andadjusted to 1ng/L. Agilent 2100 Bioanalyzer was deployed to examine the insert sizeof the acquired library. At last, the accurate concentration of cDNA library was againexamined using qPCR. Once the insert size and concentration of the library wasidentical, the samples can then be subjected for sequencing.