Hs rna screentape

Venom gland and thorax tissue samples were harvested from 94 individuals belonging to a single colony of V. affinis and snap frozen. Both of these tissue types were then homogenised separately, and the total RNA was isolated using the TRIzol™ Reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States) following the manufacturer’s protocol. DNA contamination from the extract was removed using Turbo DNase (Thermo Fisher Scientific, MA, United States), followed by the second round of extraction with the TRIzol™ Reagent. The purity and concentration of the isolated RNA samples were determined using an EPOCH 2 spectrophotometer (BioTek Instruments, Inc., United States). The integrity of the isolated RNA samples was assessed on a TapeStation system using RNA HS ScreenTape (Cat# 5067–5579; Agilent Technologies, Santa Clara, CA, United States), and samples that passed quality checks were selected for sequencing. cDNA libraries were generated using the NEBNext^® Ultra™ RNA Library Prep Kit (New England Biolabs, Ipswich, MA, United States), and sequenced on an Illumina HiSeq X platform (2 × 150 bp paired-end with a sequencing depth of 20 million reads). The raw data has been submitted to NCBI’s Sequence Read Archive (SRA) (Bioproject: PRJNA886082).

The total RNA was extracted from freshly preserved venom gland tissues of D. palaestinae using the TRIzol™ Reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. Isolated RNA samples were treated with Turbo DNase (Thermo Fisher Scientific, Waltham, MA, USA) to remove DNA contamination, followed by another round of extraction with TRIzol™. The concentration and purity of the isolated RNA were measured using an Epoch 2 microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA), and the integrity was assessed on a bioanalyzer Agilent 4200 TapeStation system using RNA HS ScreenTape (Cat# 5067-5579; Agilent Technologies, Santa Clara, CA, USA). RNA samples that had an RNA Integrity Number (RIN) of greater than 8 were used to generate cDNA library using the NEBNext^® Ultra™ RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) for Illumina^® and subsequently sequenced on an Illumina HiSeq 2500 platform, with a sequencing depth of 20 million reads (2 × 150 bp paired-end). Raw sequencing results are deposited to NCBI’s Sequence Read Archive (SRA) repository: Bioproject: PRJNA800175; SRA: SRR17903474 and SRR17903475.

Plant material for the collection of tissues for RNA sequencing (RNA-seq) and Iso-Seq was grown in the greenhouse at IPK Gatersleben with day–night temperatures of 21 °C–18 °C. Embryonic tissue, leaves, roots, internode, inflorescence (5 mm) and developing seeds (5 and 15 days after pollination) were collected as previously described^{4 (link)}, snap-frozen in liquid nitrogen and stored at −80 °C until RNA extractions were performed. RNA was extracted from the collected tissues using a Trizol extraction protocol^{4 (link)} and purified using Qiagen RNeasy miniprep columns as per the manufacturer’s instructions. RNA quality was checked on Agilent RNA HS screen tape and RNA with RIN value greater than 8 was used for RNA-seq and Iso-Seq library construction.

The NEBNext Ultra II Directional Library Prep Kit for Illumina (#NEBE7760L; New England Biolabs Inc., Ipswich, MA) was used on 25 ng of total RNA for the preparation of strand-specific sequencing libraries from each TRAP-isolated RNA sample (input, negative fraction, and positive fraction) and from conventionally isolated RNA samples from brain (tissue), according to manufacturer’s instructions. Briefly, polyA containing mRNA was purified using oligo-dT attached magnetic beads. mRNA was chemically fragmented and cDNA synthesized. For strand-specificity, the incorporation of dUTP instead of dTTP in the second strand cDNA synthesis does not allow amplification past this dUTP with the polymerase. Following cDNA synthesis, each product underwent end repair process, the addition of a single ‘A’ base, and finally ligation of adapters. The cDNA products were further purified and enriched using PCR to make the final library for sequencing. Library sizing was performed with HSRNA ScreenTape (#5067–5579; Agilent Technologies) and libraries were quantified by qPCR (Kappa Biosystems, Inc., Wilmington, MA). The libraries for each sample were pooled at 4 nM concentration and sequenced using an Illumina NovaSeq 6000 system (SP PE50bp) at the Oklahoma Medical Research Foundation Genomics Facility.

The 4150 TapeStation System (Agilent Technologies, Inc.) was used for DNA sample electrophoresis. The system uses microfluidic technology to perform CE: nucleic acid molecules migrate through a gel matrix within the tape in an electric field, and the rate of migration is determined by the size and charge of the molecules. For DNA loading, an equal volume of the loading buffer (high-sensitivity [HS] RNA loading buffer) was added, and the mixture was incubated at 75°C for 5 min. Electrophoresis was performed using an HS RNA ScreenTape (Agilent Technologies, Inc.). The quantitative range was 0.5–10 ng/μL and sizing range was 0.1–6.0 kb.