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.