RNA-seq of Helobdella austinensis: body
After acquiring stage 7 embryos, they were cultured in clean Helobdella triserialis (HTR) medium until stage 10 (220 to 245 hour after zygotic development), the period of proboscis formation. In order to obtain a sufficient amount of RNA, about 400 embryos were used in the experiment. The head region was hand-dissected using insect pins (Shiga, No. 2) under a Leica ZOOM 2000 stereomicroscope (Leica, Wetzlar, HE, Germany). Next, 30 L of HTR medium containing proboscis region was transferred to a 1.7ml tube containing 200L of TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Each sample RNA was isolated using TRIzol reagent according to the manufacturer's instructions, and the purity and integrity of the total RNA isolated from dissected embryo sample were examined using a Nanodrop 2000C spectrophotometer (Thermo Scientific, Waltham, MA, USA) and Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). In addition, the total RNA concentration was calculated using Quant-IT RiboGreen (Invitrogen, R11490). To assess the integrity of the total RNA, samples were run on TapeStation RNA screentape (Agilent, 50675576). Only high-quality RNA preparations (RNA integrity number > 7.0) were used to construct an RNA library. A library was independently prepared with 1 g of total RNA for each sample using the Illumina TruSeq Stranded Total RNA Sample Prep Kit v2 (Illumina, Inc., San Diego, CA, USA). The ribosomal RNA in total RNA was depleted using a Ribo-Zero kit. After the rRNA depletion, the remaining RNA was purified, fragmented, and primed for cDNA synthesis. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random hexamers. This was followed by second-strand cDNA synthesis using DNA Polymerase I, RNase H, and dUTP. Next, these cDNA fragments underwent end repair process, the addition of a single A base, and adapter ligation. The products were then purified and enriched with polymerase chain reaction (PCR) to create the final cDNA library. All libraries were quantified using quantitative PCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for Illumina Sequencing platforms) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, Waldbronn, BW, Germany). Finally, the resulting samples were sequenced using the Illumina HiSeq 2000 system (Illumina, Inc., San Diego, CA, USA) with a paired-end read.