Ion xpress rna seq barcode 1 16 kit

Platinum PCR SuperMix High Fidelity (45 μL) and barcode primers at 1 μL each were adjusted, 6 μL of cDNA was added, and amplification reaction was performed (a 2-min hold at 94 °C. Two cycles for the following: 30 s at 94 °C, 30 s at 50 °C, 30 s at 68 °C. Sixteen cycles for the following: 30 s at 94 °C, 30 s at 62 °C, 30 s at 68 °C, and a 5-min hold at 68 °C). Additionally, 5 μL of suspended nucleic acid binding beads and 120 μL of binding solution concentrate were mixed, and 53 μL of amplified cDNA was added. After adding 130 μL of 100% ethanol, mixing well, incubating at room temperature for 5 min and binding cDNAs to the beads, the cDNA eluate was collected as in the fragmented RNA purification procedure (However, 15 μL of nuclease-free water was added). Using Agilent 4150 TapeStation (Agilent Technologies, Santa Clara, CA, USA), library quantification was performed. In addition, the reagents used were as follows:

Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific, USA: 4475936);

IonXpress RNA-Seq Barcode 1–16 Kit (Thermo Fisher Scientific, USA: 4475485);

D1000 ScreenTape (Agilent Technologies, USA: 5067–5582);

D1000 Reagents (Agilent Technologies, USA: 5067–5583).

Total RNA was extracted from frozen tissue blocks containing $50-100$ mg of PDAC tissue or normal tissue using standard protocols. First, frozen tissues were ground and homogenized using liquid nitrogen. Total RNA was extracted using an miRNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocols. The quality, quantity, and integrity of total RNA were evaluated using a NanoDrop One/ ${\text{One}}^{\mathrm{C}}$ UV-Vis spectrophotometer (Thermo Fisher Scientific) and a Bioanalyzer 2100 (Agilent Technologies). Only samples with an RNA quality score (RIN value) $>7.0$ were used for RNA-seq. The RiboMinus Eukaryote System v2 was used to exclude rRNA from the total RNA. The mRNAs were barcoded with the Ion Xpress RNA-Seq Barcode 1–16 Kit (Thermo Fisher Scientific), and libraries were generated using the Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific). The library was constructed for next-generation sequencing (NGS) on an Ion Proton instrument (Thermo Fisher Scientific) using a $2\times 75$ bp pair-end protocol. In total, we sequenced eight libraries, generating 34–60 million pairs of reads per sample. NGS BAM files containing the sequence data were then analyzed by bioinformaticians. The number of reads mapped to annotated genomic features was quantified from the BAM files using the feature counts in the Subread package. The sample data were normalized to count per million (CPM).

A pool of five Anopheles sacharovi individuals were homogenized and total RNA was extracted by TRIzol reagent (Thermo Fischer Scientific) according to the manufacturer’s protocol. Whole transcriptome libraries were prepared from 500 ng of RNA extract, using the Ion Total RNA-Seq v2 Core Kit (#4479789, ThermoFisher Scientific) according to the manufacturer’s instruction. In brief, the RNA library preparation involved RNA fragmentation, adapter ligation, reverse transcription and 14 cycles of PCR amplification using Ion Xpress RNA-Seq Barcode 1–16 Kit (#4475485, ThermoFisher Scientific). Quantification of the library was performed using Qubit Fluorometer high-sensitivity kit (ThermoFisher Scientific) and its median size was determined in LabChip GX Touch 24 (PerkinElmer). The libraries were loaded onto an Ion 540 chip, using Ion Chef (Thermo Fisher Scientific) and sequencing was carried out on an Ion GeneStudio S5 sequencer (ThermoFisher Scientific). Ion GeneStudio S5 sequencer returns the reads already quality trimmed.