title: RNA-Seq of ANAMMOX and FEAMMOX bacteria
design: The DNA was extracted using DNeasy Powersoil kit and quantified using PicoGreen.The V3 and V4 regions of the bacterial 16S rRNA gene, which was devised to  detect microbial community at the species level, were amplified with universal primers SD182 Bact-0341-bS-17 (5'-CCTACGGGNGGCWGCAG-3') and SD-Bact-0785-aA-21 (5'- GACTACHVGGGTATCTAATCC-3'). The Illumina 16S Metagenomic Sequencing Library  protocol was used to construct the bacterial DNA library (Macrogen, Korea). Polymerase chain reaction (PCR) amplification was performed with 2 ng genomic DNA using the Herculase II  fusion DNA Polymerase Kit (Agilent Technologies, US). The procedure followed the  instruction manual. Amplicons were purified using AMPure beads (Agencourt Bioscience, US).  For final library generation, 1 L of amplicons was used for nested PCR amplification. After  purification, samples were prepared using the qPCR Quantification Protocol Guide (KAPA  Library Quantification kits for Illumina sequencing platforms), and gel electrophoresis was performed using a TapeStation D1000 ScreenTape (Agilent Technologies). Paired-end (2300  bp) sequencing was performed using the MiSeq platform (Illumina, US). After complete  sequencing, the MiSeq raw data were classified by sample using the index sequence, and a  FASTA file was created for each sample.