Metatranscriptomes of activatied sludges
Total RNA was extracted from floccular and granular biomass using a RNA Clean and ConcentratorTM-5 kit (ZYMO Research Corporation, Singapore) according to the manufacturerŐs guidelines. DNA was removed using TURBO DNA-free kit (Applied Biosystems, Singapore). RNA quality was determined using Agilent 2100 Bioanalyser and reported as RNA integrity number. The quantity of total RNA and residual DNA was measured by Quant-iT RiboGreen RNA and PicoGreen DNA assays (Invitrogen, Singapore), respectively. Total RNA, 200 ng, was used for complimentary DNA (cDNA) library preparation according to the manufacturerŐs instructions (Illumina, Singapore). Each cDNA library was ligated with a unique adaptor sequence for sample multiplexing. A total of 12 different cDNA libraries were pooled and sequenced by Illumina Hiseq (Hiseq 2500, Illumina) and a total of 263 million raw sequencing reads with an average read length of 101 nucleotides were generated.