Tbs380 picogreen

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).

Total RNA from all the samples was extracted using the Trizol (produced by Invitrogen) basing on the manual. Then the total RNA was purified by magnetic stand (Invitrogen). The Aliquots of total RNA purified were stored in the − 80 °C refrigerator. The libraries of sequencing were constructed by 5 μg total RNA using the TruSeq™ RNA sample preparation Kit (Illumina Inc., San Diego, USA) following the instructions of manufacturer. According to the protocol of library construction (Illumina), synthetic cDNA was treated with end-repair, phosphorylation and ‘A’ base addition. After PCR treated by NEB’s Phusion DNA polymerase for 15 cycles, selection of size was performed for target fragments of cDNA on 2% Agarose of Low Range Ultra (Bio-Rad). The size of cDNA target fragments is 200–300 bp. Then the libraries were quantitated with TBS380 Picogreen (Invitrogen). All libraries of paired-end sequencing were sequenced using the HiSeq xten (2 × 150 bp read length) (Illumina Inc., San Diego, USA).

Total RNA was extracted from the samples with TRIzol® Reagent (Invitrogen) according to the kit’s instructions. The total RNA concentrations and quality in the tissue samples were then assessed by NanoDrop2000 and Agilent 2100. Specifically, OD260/OD280 ratios between 1.8 and 2.2, and OD260/OD230 ratios of greater than 1.8 deemed acceptable, while RIN should be over 0.85. In order to enrich pure circRNAs, rRNAs and linear RNAs were removed from the total RNA in each sample by Ribo-Zero Magnetic kit and RNase R (Epicentre) treatment. RNA-seq library were preparared by using the TruSeq™ Stranded Total RNA Library Prep Kit (Illumina) following the manufacturer’s instructions. (LifeTechnologies,USA). TBS380 Picogreen (invitrogen) was adopted to quantified the libraries and Illumina Hiseq. 4000 were used for pair-end HTS. The library preparation and sequencing was performed at Major-Biotech Inc. (Shanghai, China).

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 was isolated from the WT and NOD1-1IS^−/− zebrafish at 10 dpf using the TRIzol® Reagent (Invitrogen). The quantity and quality of total RNA were checked by Nanodrop2000 (Thermo Scientific) and Bioanalyzer 2100 (Agilent). Sequencing libraries for whole transcriptome analysis were generated using TruseqTM RNA sample prep Kit (Illumina). After quantitation using TBS380 Picogreen (Invitrogen), the libraries were sequenced using Hiseq4000 Truseq SBS Kit v3-HS(200cycles)(Illumina). To identify differentially expressed genes between the WT and NOD1-1IS^−/− zebrafish, the expression levels were measured by using numbers of fragments per kilobase of transcript per million fragments sequenced (FPKM). The raw sequences were deposited at NCBI Sequence Read Archive (Accession No. SRP090717).

Total RNA was extracted from 40–50 larvae by Trizol reagent (Invitrogen, Carlsbad, CA). The RNA quality was checked by Bioanalyzer 2200 (Aligent, Santa Clara, CA) and kept at -80°C. miRNAs were purified by miRNeasy Mini Kit (Qiagen, Germany) from RNAs with RIN > 8.0. The cDNA libraries for single-end sequencing were prepared using Truseq^™ Small RNA sample prep Kit (Illumina, San Diego, CA) according to the manufacturer’s instructions. 1 μg small RNA was connected to add 3’ and 5’ adaptors and then enriched by PCR. Size selection was applied to obtain the 145–160 bp library sequence. TBS-380 Picogreen (Invitrogen, Carlsbad, CA) was introduced to quantify the library concentration. The sequencing was performed on Hiseq2000 platform (Illumina, San Diego, CA).