Ribominus eukaryote kit for rna seq

Total RNA samples were isolated using the RNeasy mini kit (QIAGEN #74106) including on-column DNase treatment. mRNA was purified from 10 to 50 μg of total RNA by two cycles of poly-A enrichment using the Oligotex kit (QIAGEN #72022) followed by an rRNA removal step using the RiboMinus Eukaryote Kit for RNA-Seq (Invitrogen #A1083702). Precipitated mRNA samples were eluted with 9 ml of RNase-free water and fragmented with 1 ml of fragmentation buffer (Ambion, #AM8740) at 70 °C. Reactions were stopped after 5 min by adding 1-ml stop buffer, and RNA was purified by ethanol precipitation. cDNA was synthesized from 100 to 300 ng of mRNA using SuperScript III reverse transcriptase (Invitrogen #18080-051). Double-stranded DNA was synthesized according to the instructions using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen). DNA was cleaned with a QIAquick PCR spin column (QIAGEN, #28106), sequencing adapters were ligated according to the Illumina protocol, and the library was amplified by 18 cycles of PCR using Phusion DNA polymerase (NEB, #F-530). Bands around 300 bp were gel purified and libraries were sequenced on the Illumina HiSeq 2000 platform at the Vincent J. Coates Genomic Sequencing Laboratory at UC Berkeley to generate single-end 100 base reads.

Total RNAs extracted from tick homogenates positive for flavivirus were used for whole genome sequencing. Ribosomal RNA depletion from total RNA was performed using RiboMinus Eukaryote Kit for RNA-Seq (Invitrogen), and cDNA was synthesized using a PrimeScript Double Strand cDNA Synthesis Kit (Takara) according to the manufacturers’ instructions. The cDNA libraries were prepared using a Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) according to the manufacturer’s instructions, and were then subjected to whole-genome sequencing on a MiSeq using a MiSeq Reagent Kit v3 (600 cycles) (Illumina). Sequencing data was analyzed using the CLC Genomics Workbench 12.0 (https://digitalinsights.qiagen.com; CLC bio, Hilden, Germany). Flavivirus genome contigs were obtained by de novo assembly and the overlapped contig sequences were confirmed by PCR amplification with specific primers and Sanger sequencing. The 5′ and 3′ termini of the flavivirus genome were amplified using RACE with specific primers and a SMARTer RACE cDNA Amplification Kit (Takara) according to the manufacturer’s protocol (see Supplementary Table S2 online). Amplified products were directly sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit.

DENV2-infected and uninfected blood fed control mosquitoes were dissected at 8 dpbm, and 100 midguts for each condition were pooled and stored in TRIzol reagent (Invitrogen). Total RNA from mosquito midguts was extracted using TRIzol and rRNA was removed by hybridization using RiboMinus Eukaryote Kit for RNA-Seq (Invitrogen). Polyadenylated mRNA was then isolated from mosquito midguts by one round of selection with the Dynabeads mRNA Purification Kit (Invitrogen). Quality of mRNA was assessed by electrophoresis on the Bioanalyzer 2100 (Agilent). For RNAseq sample preparation, NEBNext mRNA Sample Prep Master Mix Set 1 was used according to the manufacturer’s protocol (NEB). Briefly, 0.5ug mRNA was used for fragmentation and then subjected to cDNA synthesis using SuperScript III Reverse Transcriptase (Invitrogen) and random primers. The cDNA was further converted into double stranded cDNA and, after an end repair process (Klenow fragment, T4 polynucleotide kinase and T4 polymerase), was ligated to Illumina paired end (PE) adaptors. Size selection was performed using a 2% agarose gel, generating cDNA libraries ranging in size from 275–325bp. Finally, the libraries were enriched using 15 cycles of PCR and purified by the QIAquick PCR purification kit (Qiagen).

Libraries were prepared as described before [47 (link)]. Yeast cells were grown to OD₆₀₀ = 0.4–0.8 in YPD medium. Total RNA was extracted using the hot phenol extraction procedure. Ribosomal RNAs were depleted from 10 μg of total RNA using the RiboMinus™ Eukaryote Kit for RNA-Seq (Invitrogen). Quality of total and rRNA-depleted RNA was checked by Northern blot and with a RNA Pico 6000 chip in an Agilent 2100 Bioanalyzer. RNase III fragmentation was performed according to the SOLiD^® Total RNA-Seq Kit protocol (Applied Biosystems, Life Technologies, Cergy Pontoise, France), starting from 750 ng of rRNA-depleted RNA. Fragmented RNA were cleaned up using the RiboMinus™ Concentration Module (Invitrogen) and eluted in 20 μL of nuclease-free water. Running a sample of fragmented RNA on a RNA Pico 6000 chip in an Agilent 2100 Bioanalyzer checked RNA fragmentation. RNA-Seq libraries were constructed using the SOLiD^® Total RNA-Seq Kit (Applied Biosystems) according manufacturer’s instruction, starting from 60 ng of fragmented RNA.

After each respective culture time (SPO3H, SPE3H, SPO12H and, SPE12H), parasites were collected and washed twice with MEM-HEPES medium and immediately submitted to RNA extraction. Total RNA extraction and isolation was performed with TRIzol Reagent (Invitrogen, cat15596-026) and, RNeasy (Qiagen, cat74106), with a final treatment with DNAse Turbo (Ambion, catAM1907), all according to the procedures recommended by manufactures. The rRNA was depleted using RiboMinus Eukaryote Kit for RNAseq (Invitrogen, cat10837-08). Before rRNA depletion, mRNA quality was verified by measuring rRNA integrity through capillary electrophoresis in a Bioanalyzer 2100 (Agilent, catG2939AA), employing RNA 6000 Pico Reagents (Agilent, cat5067-1514). Quantitation of mRNA was assessed by using a Qubit 2.0 Fluorometer (Life Technologies catQ32866) and a Qubit RNA Assay Kit (Invitrogen catQ32852) for RNA samples, according to the protocol recommended by the manufacturer.

Total RNAs, which were obtained from Asian seabass testes and ovaries, were depleted of ribosomal RNA using a RiboMinus Eukaryote Kit for RNA-seq (Invitrogen) and verified using an Agilent 2100 Bioanalyzer. The rRNA-depleted total RNA was sent to a service provider for transcriptome sequencing on a SOLiD 3+ platform (Applied Biosystems). Similarly, another rRNA-depleted sample, which was pooled from RNA that was extracted from various Asian seabass organs, was sent to another service provider for transcriptome sequencing on a 454 FLX Titanium platform (Roche). Reads that were obtained from SOLiD 3+ and 454 sequencing were assembled de novo using the programs Velvet, CLC Genomics Workbench (CLC Bio) and Sequencher (Gene Codes)
[35 (link)]. The reads and assembled transcriptome have been deposited into the NCBI SRA and TSA databases, respectively [SRA accession numbers SRR944005, SRR944006 and SRR949061; TSA accession numbers GAML01000000 and GAMU01000000].