Miseq v3 sequencing kit

Fecal concentrations of EDP1066 for stool persistence and prevalence and the gut microbiome were measured by Diversigen Inc. (Houston, TX, USA) using validated bioanalytical assay methods. In short, fecal microbial DNA was extracted based on the Zymo Research (Irvine, CA, USA) fecal DNA extraction methodology. EDP1066-specific primers and probes had been developed to enable the detection of the L. lactis spp. cremoris strain. The fecal samples were analyzed using a qPCR with a lower limit of quantification of 5.0 copies/5 ng DNA. For gut microbiome analyses, extracted DNA was prepared for Illumina sequencing via PCR amplification of the variable region 4 of the bacterial 16S rRNA gene. After PCR purification using AMPure XP beads (Beckman Coulter Life Sciences, Indianapolis, IN, USA), sample-specific barcodes using Illumina Nextera XT Index kit (Illumina Inc., San Diego, CA, USA) were appended to the PCR products during a second PCR. The PCR products were purified for a second time, and lastly, the PCR products were equimolarly pooled and sequenced on the Illumina MiSeq platform using the MiSeq v3 sequencing kit.

We collected three pairs of forelegs from 0-, 1-, 3-, and 5-day-old females. Also, a pair of antennae from a 0-day-old female was also collected. Total RNA samples were extracted using QuickGene RNA tissue kit SII (Kurabo, Osaka, Japan). cDNA library preparation was carried out using TruSeq RNA Sample Prep Kit v2 (Illumina, CA, USA) following the manufacturer's protocol with a modification of the incubation time for RNA fragmentation. To obtain longer fragments, we set the incubation time to 1 min. RNA-sequencing runs were performed using MiSeq system with MiSeq sequencing kit v3 (Illumina). Raw RNA-sequencing data have been deposited in the DNA Data Bank of Japan (DDBJ) Sequence Data Archive under accession numbers DRA011862 and DRA011865. Sequenced reads were assembled using Trinity 2.0.6 and 2.8.4 for antenna samples and foreleg samples, respectively, with Quality Trimming Options by Trimmomatic (LEADING: 10; TRAILING: 10; SLIDINGWINDOW: 4:20; MINLEN: 150). Kmer_size parameter was set at 32. Coding regions and amino acid sequences of the assembled contigs were predicted using TransDecoder.