Miseq 500 cycles reagent kit v2

For microbial community structure analysis of the UASB samples, the V4 regions of the 16S rRNA gene were amplified using fusion primers for the Illumina sequencing platform (Caporaso et al. 2012 (link)). Multiplex identifiers of 12 nucleotides were incorporated in the M806R primer to allow for multiplexing. PCR conditions were described elsewhere (Lu et al. 2006 (link)). Following amplification, PCR products for each sample were purified using the Agencourt AMPure XP PCR purification system (Beckman-Coultier) and quantified using the Qubit Fluorometer (Invitrogen). Amplicons were sequenced from both the forward and reverse primers using the Illumina MiSeq platform and the MiSeq 500 cycles reagent kit v2 (Illumina Inc.). Sequence data generated were demultiplexed and processed using the QIIME pipeline (Caporaso et al. 2010 (link)). Sequences were clustered at 97% sequence identity, and the taxonomy of the representatives from each OTU was assigned using blastn (Camacho et al. 2009 (link)) against the Greengenes database (DeSantis et al. 2006 (link)).

The cleavage sequence was identified using the protocols described in our previous study [25 (link)]. First, 1.5 μg of five RNA mixtures were incubated with 400 ng of MazF_NE1181 at 37 °C for 30 min in MazF reaction buffer. Phosphorylation, barcode ligation, and sequencing library construction were performed as described by Miyamoto et al. [25 (link)]. Sequencing was performed using the MiSeq platform with the MiSeq 500 cycles reagent kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. Sequence data was analyzed using CLC Genomics 7.5.1. The parameters described by Miyamoto et al. [25 (link)] were used for the analysis, and 25 sequences were analyzed using WebLogo [48 (link)]. The deep sequencing dataset was deposited into the DDBJ Sequence Read Archive (DRA004562).