Dynabeads oligo dt 25 mrna purification kit

Genomic DNA for genome assembly and Illumina sequencing was extracted from a single male pupa by CTAB extraction^{63 (link)}. High molecular weight DNA for Nanopore sequencing was extracted from three male pupae using the MagAttract HMW DNA kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
For analysis of silk-related genes, total RNA from last larval instar silk glands was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA), followed by isolation of mRNA using Dynabeads Oligo (dT)25 mRNA Purification Kit (Thermo Fisher Scientific, Waltham, USA), and cDNA was prepared using the NEXTflex Rapid RNA-Seq Kit (Bioo Scientific, Austin, USA). Additionally, to annotate the genome, RNA from heads, thoraces, and gonads of three male and female imagoes was extracted with TRI-Reagent (Sigma-Aldrich) according to the provided protocol. Biological replicas were pooled prior to isolation, resulting in three tissue-specific samples per each sex.

RNA isolation, synthesis of cDNA libraries and RNA sequencing were performed as previously described [28 (link)]. Total RNA from the dissected larval silk glands of P. prasinana was isolated using Trizol reagent (Life Technologies, Carlsbad, CA, USA) and used to prepare cDNA libraries for the Illumina sequencing platform. rRNA was removed using “A RiboMinus Eukaryote Kit for RNA-Seq” (Ambion, Austin, TX, USA). poly-A mRNA was enriched using a Dynabeads Oligo (dT)₂₅ mRNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA), and the cDNA library was prepared with a NEXTflex Rapid RNA-Seq Kit (Bioo Scientific, Austin, TX, USA). Sequencing was performed using a MiSeq instrument (Illumina, San Diego, CA, USA), producing sequences in the 2 × 150 nt pair-end format. A total of 1.6 × 10⁷ reads were assembled de novo using Trinity software (v.2.9.1) on the Galaxy platform [29 (link)] set on the default options and a minimum allowed length of 200 bp. The transcriptome database served for manual searches of homologs of known silk proteins from other species by local BLAST in BioEdit (v.7.2) software [30 ] and also as a backbone for the identification of tryptic peptides in MS spectrometry. The nucleotide sequences were uploaded to NCBI GenBank under the codes MW373748–MW373775.