Truseq rna sample preparation kit v2 set a

Using the TruSeq Small RNA Sample Prep Kit (Illumina), total RNA, containing small RNAs, was reverse transcribed into a cDNA library. In brief, 1 µg of total RNA per sample was ligated with 5' and 3' adapters, reverse transcribed, treated with RNase, and amplified with PCR using specific labeled amplification primers. To select the size of small RNAs, samples were migrated on 6% native polyacrylamide gels. To collect miRNA populations, cDNA fragments between 145 and 160 bp were cut from the gel, eluted and precipitated. Finally, cDNA chip was dried and suspended in 10 µL of nuclease free water. In parallel, cDNA library was built from total RNA (1 µg per sample) from the same samples. Purification and fragmentation were performed using the TruSeq RNA Sample Preparation Kit v2 - Set A (Illumina). Next, cDNA samples were end repaired using End Repair Mix reagent (Illumina) and double-stranded cDNAs were purified and enriched to create the cDNA library. The quality of cDNA in each final miRNA and mRNA libraries was assessed using the Agilent 4200 TapeStation System (Agilent) and the amount was verified using Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen). Equimolar concentrations of each final library were pooled at a final concentration of 2 nM of cDNA. Libraries were run on the Illumina NextSeq 500 platform using the High Output kit v2 (75 cycles, Illumina).

Total RNA was extracted from six 12th larval head, thoracic, and abdominal tegument using TRIzol reagent (Ambion) and the RNeasy Plus Mini Kit (No. 74134; Qiagen, Hilden, Germany), following the instructions of the manufacturer, respectively. RNA quantity was detected using the NanoDrop 8000 (Thermo, Waltham, MA, USA). There is one replication, each replication with six larvae. RNA of 12th larval heads and thoracic and abdominal tegument were used to construct the cDNA libraries. cDNA library construction and Illumina sequencing of samples were performed at MajorBioCorporation (Shanghai, China). mRNA samples were purified and fragmented using the TruSeq RNA Sample Preparation Kit v2-Set A (No. RS-122-2001; Illumina, San Diego, CA, USA). Random hexamer primers were used to synthesize the first-strand cDNA, followed by synthesis of the second-strand cDNA using a buffer, dNTPs, RNase H, and DNA polymerase I at 16°C for 1 h. After end repair, A-tailing, and the ligation of adaptors, the products were amplified by PCR and quantified precisely using the Qubit DNA Br Assay Kit (Q10211; Invitrogen, Carlsbad, CA, USA). They were then purified using the MinElute Gel Extraction Kit (Qiagen, Cat No. 28604) to obtain a cDNA library. The cDNA library was sequenced on the HiSeq2500 platform.

Total RNA was extracted from two female and two male antennae using TRIzol reagent (Ambion) and the RNeasy Plus Mini Kit (No. 74134; Qiagen, Hilden, Germany) following the manufacturer’s instructions. RNA quantity was detected using the NanoDrop 8000 (Thermo, Waltham, MA, USA). A quarter of the total RNA for the antennae of each male and female was mixed and used to construct the cDNA library. The cDNA library construction and Illumina sequencing of samples were performed at CapitalBio Corporation (Beijing, China). The mRNA samples were purified and fragmented using the TruSeq RNA Sample Preparation Kit v2-Set A (No. RS-122-2001; Illumina, San Diego, CA, USA). Random hexamer primers were used to synthesize the first-strand cDNA, followed by synthesis of the second-strand cDNA using buffer, dNTPs, RNase H, and DNA polymerase I at 16 °C for 1 h. After end repair, A-tailing, and the ligation of adaptors, the products were amplified by PCR and quantified precisely using the Qubit DNA Br Assay Kit (Q10211; Invitrogen, Carlsbad, CA, USA). They were then purified using the MinElute Gel Extraction Kit (Qiagen, Cat No. 28604) to obtain a cDNA library. The cDNA library was sequenced on the HiSeq2000 platform.

Total RNA was extracted from two female and two male antennae using TRIzol reagent (Ambion) and the RNeasy Plus Mini Kit (No. 74134; Qiagen, Hilden, Germany) following the manufacturer’s instructions. RNA quantity was detected using the NanoDrop 8000 (Thermo, Waltham, MA, USA). RNA of male and female antennae was used to construct the cDNA library respectively. cDNA library construction and Illumina sequencing of samples were performed at CapitalBio Corporation (Beijing, China). mRNA samples were purified and fragmented using the TruSeq RNA Sample Preparation Kit v2-Set A (No. RS-122-2001; Illumina, San Diego, CA, USA). Random hexamer primers were used to synthesize the first-strand cDNA, followed by synthesis of the second-strand cDNA using buffer, dNTPs, RNase H, and DNA polymerase I at 16 °C for 1 h. After end repair, A-tailing, and the ligation of adaptors, the products were amplified by PCR and quantified precisely using the Qubit DNA Br Assay Kit (Q10211; Invitrogen, Carlsbad, CA, USA). They were then purified using the MinElute Gel Extraction Kit (Qiagen, Cat No. 28604) to obtain a cDNA library. The cDNA library was sequenced on the HiSeq2500 platform.

We extracted total RNA from whole body of C. obducta males and females utilization TRIzol reagent (Ambion, Austin, TX, USA) and the RNeasy Plus Mini Kit (No. 74134; Qiagen, Hilden, Germany) according to the manufacturer’s instructions. NanoDrop2008 (Thermo, Waltham, MA, USA) and agarose gel electrophoresis examined density and quality of RNA. Half RNA of male and female bodies with three biological replicates were used to construct three cDNA libraries respectively. Construction cDNA libraries and Illumina sequencing of samples were implemented at Majorbio Corporation (Shanghai, China). Using TruSeq RNA Sample Preparation Kit v2-Set A (No. RS-122-2001; Illumina, San Diego, CA, USA) was to perform purification and fragmentation of mRNA samples. The first-strand cDNA was synthesized by utilization random hexamer primers, then using RNase H, dNTPs, buffer and DNA polymerase I at 16 °C for 1 h to synthesize the second-strand cDNA. After end repair, A-tailing and the ligation of adaptors, the products were amplified by PCR and quantified precisely by the Qubit DNA Br Assay Kit (Q10211; Invitrogen, Carlsbad, CA, USA). cDNA libraries were obtained after they were purified by the MinElute Gel Extraction Kit (Cat No. 28604; Qiagen, Hilden, Germany). On the HiSeq2500 platform three cDNA libraries were sequenced.