title: De novo transcriptomic response of Pirenella cingulata (Gastropoda) infected with trematode parasite
design: RNA was extracted from snail tissue using Qiagen RNeasy Mini Kit (Cat# 169045471) and quantified using Qubit RNA BR Assay (Invitrogen, Cat# Q10211). RNA purity was checked using QIAxpert and RNA integrity was checked on TapeStation using RNA Screen Tapes (Agilent, Cat# 50675579). Library was prepared using manufactures protocol of NEB Ultra II directional RNA-Seq Library Prep kit (cat #E7760L). Total RNA of 500ng concentration was taken for the assay. magnetic Poly(T) beads (NEB, Cat# E7490L) was used to capture mRNA. The purification was followed by mRNA fragmentation using divalent cations under increased temperatures. Reverse transcriptase was utilized to transform the cleaved RNA fragments into first-strand cDNA. The enzymes RNase H and DNA Polymerase I were used to synthesize second-strand cDNA. Afterward, the cDNA fragments underwent a sequence of enzymatic processes that repaired the ends, tailed the 3&#39; end with a single &quot;A&quot; base, and finally ligated the adapters. The following thermal conditions were then used to purify and enrich the adapter-ligated products: 98C for 30 seconds of initial denaturation; 12 cycles of -98C for 10 seconds, 65C for 75 seconds; and a final extension of 65C for 5 minutes. After that, PCR products are purified and their fragment size distribution is examined using an Agilent HS NGS Fragment Kit (1-6000bp) (Cat# DNF-474-1000) on a fragment analyzer. 6 Prepared libraries were quantified using Qubit HS Assay (Invitrogen, Cat# Q32854). The obtained libraries were pooled and diluted to the final optimal loading concentration. The pooled libraries were loaded onto the Illumina Nova Seq V1.5 instrument to generate 30M, 150bp paired-end reads/sample.