Certified low range ultra agarose

The TruseqTM RNA Sample Prep Kit (Illumina, USA) was used to prepare the RNA-seq
transcriptome libraries. DNA-free mRNA was captured by magnetic Oligo (dT) beads
(Invitrogen) and fragmented to a size of 200 bp with a fragmentation buffer.
Using mRNA as a template, double-stranded cDNA was synthesized with a
SuperScript double-stranded cDNA Synthesis Kit (Invitrogen) using random hexamer
primers (Illumina). Then the end fragments were subjected to end-repair and `A’
base addition. PCR amplification was carried out for 15 PCR cycles. Then, cDNA
target fragments were selected on 2% Certified Low Range Ultra Agarose (Bio-Rad,
USA) and quantitated by TBS380 Picogreen (Invitrogen).
The clustering of the index-coded samples was performed on a cBot Cluster
Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina)
according to the manufacturer’s instructions. After cluster generation, the
library preparations were sequenced on an Illumina Hiseq4000 Truseq SBS Kit
v3-HS (200 cycles).

The mRNA of each sample’s three biological replicates were respectively enriched using cellulose oligo (dT) magnetic beads (Invitrogen, USA), then fragmented into ca. 200-bp fragments. The fragments were transcribed and double-stranded cDNA was synthesized, then end repair, 3′-end single-nucleotide A (adenine) addition and ligation of adaptors were performed according to the manufacturer’s instructions. The resultant fragments were enriched by PCR and purified using 2% Certified Low Range Ultra Agarose (Bio-Rad) to create the final cDNA library. The quantitative assay was conducted using Picogreen fluorescent dye (Invitrogen, USA) with a TBS-380 fluorimeter (Invitrogen, USA). After bridge PCR amplification on Illumina cBot using Truseq PE Cluster Kit v3-cBot-HS, paired-end (2 × 151 bp) sequencing of the cDNA library products was carried out using the Illumina HiSeq4000 platform.

A single mosquito was placed in a 1.5 mL centrifuge tube and was thoroughly ground after adding liquid nitrogen. Then, the adult mosquito genomic DNA was extracted using a DNeasy Blood Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The genomic DNA concentration of the extracted samples was measured by the Qubit method, and OD₂₆₀/OD₂₈₀ was detected by Nanodrop (Thermo Scientific, USA). Genomic DNA samples were stored at −80 °C for further procedures. The DNA was broken into 300–500 bp fragments by the instrument Covaris M220 (Covaris, Woburn, MA, USA). Libraries were then constructed using a TruSeq RNA sample Prep Kit (Illumina, San Diego, CA, USA) and quantified using TBS380 Picogreen (Invitrogen, Carlsbad, CA, USA) reagent. Subsequently, the library was recovered using Certified Low Range Ultra Agarose (Bio-Rad, Richmond, VA, USA). The products were amplified and enriched using a cBot TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, USA) reagent through PCR reaction, and the cycling conditions were 95 °C for 3 min, followed by 8 cycles of 98 °C/20 s, 60 °C/15 s, 72 °C/30 s, with a final extension at 72 °C for 5 min. Finally, the products obtained were sequenced using a TruSeq SBS Kit (Illumina, San Diego, USA) and in a run of 300 cycles in Illumina Novaseq platform (Illumina, San Diego, USA).

Total RNA of collected specimens was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manual instruction. Firstly, total RNA was subjected to ribosomal RNA removal by Ribo-Zero Magnetic kit (EpiCentre, Mandison, WI, USA); secondly, cDNA library was constructed by Truseq RNA sample Prep Kit (Illumina, Inc., San Diego, CA, USA). RNA was purified and fragmented into 200 base pairs; RNA fragment primed with random hexamer primers were applied for the first cDNA strand synthesis; then the second cDNA strand was synthesized and dUTP was used instead of dTTP; the double cDNA strands were underwent end pair, 3′end adenylation and adapter ligation; the second cDNA strand was digested by UNG enzyme (Illumina, Inc., San Diego, CA, USA). Thirdly, libraries were amplified through polymerase chain reaction for 15 cycles, purified through Certified Low Range Ultra Agarose (Bio-Rad) and quantified through Picogreen (Molecular probes) on TBS380 (Turner Biosystems). Then bridge PCR was performed on cBot. Lastly, each library was loaded into one lane of the Illumina HiSeq 4000 for sequencing.

Chinese salamanders were obtained from Zhoushan Island (Zhejiang, China), Zhoushan Island is the largest island of Zhoushan Archipelago. Total RNA was extracted from the whole body of four Chinese salamanders using Trizol Reagent (Invitrogen) according to the manufacturer’s instruction. Ahead of cDNA library construction, the total RNA was treated with DNase I, and magnetic beads with Oligo (dT) were used to enrich poly (A) mRNA from it. Then, the purified mRNA was disrupted into short fragments and the double-stranded cDNA was synthesized and subjected to end-repair, add poly (A) and connect with sequencing adapters using Truseq™RNA sample prep Kit (Illumina). The suitable fragments purified by 2% agarose gel electrophoresis (Certified Low Range Ultra Agarose, Bio-Rad) were selected as templates for PCR amplification. Finally, the library was sequenced using Illumina HiSeq™ 2000.

Biofilm cells were grown for 24-h in TSB OR in pH 10 medium and were harvested by centrifugation (4000 rpm, 15 min) at 4°C. Cells were then resuspended with TRIzol (Invitrogen, Carlsbad, USA) and disrupted with a Mini-Bead beater (Biospec, California, USA). Total RNA was isolated according to the manufacturer's instructions for TRIzol-chloroform extraction (Invitrogen, California, USA). Genomic DNA was removed with Turbo DNase. Ribosomal RNA (rRNA) was removed with a Ribo-Zero Magnetic kit (G+/G-Bacteria) (Epicentre, Wisconsin, USA). The cDNA library was constructed with extracted mRNA according to a TruseqTM RNA sample prep kit (Illumina, California, USA). The sample was purified and ligated to the genomic adapters provided by Illumina. The purified samples were amplified by 15-cycle PCR. After ligation, the sample was loaded on a 2% agarose E-gel (Invitrogen). The targeted fragments were excised from the gel and purified using Certified Low Range Ultra Agarose (Bio-Rad). The sample was quantified using TBS380 Picogreen (Invitrogen). Sequencing was carried out on the Illumina HiSeq 2000 platform (Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China).