RNA extraction, sequencing and data processing
Total RNA was extracted from the head and abdomen of each individual separately by homogenizing each tissue in TRIzol (Thermo Fisher Scientific), then purified using Direct-zol RNA MiniPrep kits (Zymo Research) following manufacturer’s recommended protocol. Small non-coding RNA (sncRNA) library preparation and sequencing was done by the Swedish National Genomics Infrastructure. Libraries were constructed using Illumina TruSeq small RNA library kits and then sequenced using Illumina HiSeq2500 50SR. Once libraries were prepared, each sample was split and sequenced in two separate lanes to prevent any lane bias in sequence count.
The two raw sequence files for each sample were merged together prior to data processing. The tool miRTrace (Kang et al., 2018) was used for quality filtering (i) removing reads with a PHRED score below 20, (ii) removing adapters, (iii) removing reads with highly repetitive or ambiguous nucleotides, and (iv) trimming reads by length (removing reads shorter than 18nt); default settings were used with Bombyx morias a reference.