DOI: 10.6084/m9.figshare.21922752
title: Raw and mapped small RNA seq reads post oral delivery of dsRNAs to Psylliodes chrysocephala

description: Important note: Please contact the corresponding authors if you want to use the small RNA seq reads for a purpose other than what was used in the original paper, i.e., mapping the reads onto the delivered dsRNA sequences or in case of any other questions. <br> Email: doga.cedden@biologie.uni-goettingen.de <br> Raw and trimmed small RNA seq reads obtained from <em>Psylliodes chrysocephala </em>treated with double-stranded (ds) RNAs (dsSec23, dsVatpG, or dsGPF). The".bam" files are the output of Bowtie2, representing the coverage of reads perfectly mapping onto the respective dsRNA treatment. "_reads" files are the raw reads obtained by sequencing on a DNBSEQ-G400 platform and the "_trimmed.fq.gz" files represent the reads cleaned from adaptor sequences as well as reads having quality scores below 30 and lengths below 18 nt and above 25. The bam files could be investigated by, for instance, R package "viRome" which can output the coverages per position on the respective dsRNA sequence (see viROME code example.R file for the code used in our paper ) or directly using a software such as Tablet for which the dsRNA index files are required and also provided here ("_index" files). Of note the dsRNA sequence in the index file dsVatpG_index is antisense of its target gene whereas for dsSec23_index it is in sense direction. <br> Further details regarding the methodology used during the sampling and sequencing is as follows: We performed small RNA sequencing to investigate siRNAs generated from the delivered dsRNAs. The whole bodies of three male and three female pre-aestivation beetles that had received the 200 ng/cm2 <em>Sec23</em>, <em>VatpG</em>, or GFP dsRNA treatment were sampled between 2 DPT and 3 DPT by placing them in liquid nitrogen (n = 3). Specifically, beetles that had consumed at least 50% of the dsRNA-containing leaf disk were selected. The total RNA from the beetles was extracted using the Quick-RNA Tissue/Insect Kit (Zymo Research EUROPE GMBH, Freiburg, Germany) and the RNA integrity was checked via RNA fragment analysis using Agilent 2100 Bioanalyzer (Agilent Technologies). The NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (New England Biolabs GmbH, Frankfurt, Germany) kit was used for the library preparation according to the manufacturers’ instructions and all libraries were pooled based on their concentrations (see Tab. S5 for indexing information). The cDNAs within the range of 141 to 172 bp were size‑selected through gel electrophoresis, and a final concentration of 6.4 ng/µL was obtained. The small RNA sequencing was performed by BGI Tech Solutions Co. Ltd. (Hong Kong) on a DNBSEQ-G400 platform. The number and quality of the raw reads were checked using FastQC (v0.11.9) (Tab. S6). The adapters and low-quality reads were removed from the raw reads using Trim Galore! (v0.6.7), while reads within the range of 18-25 nt were retained. Cleaned and filtered reads were mapped onto the dsRNA sequences via Bowtie2 (v2.4.5) using the very sensitive end‑to-end alignment option and allowing zero mismatches. The small RNA reads from GFP dsRNA treated beetles were mapped onto <em>Sec23</em> and <em>VatpG</em> dsRNA sequences as background control.<br>