Cleantag small rna library prep kit

EVs were purified by centrifugation at 100,000× g for 1.5 h at 4 °C. The pellet was lysed with 1000 µL Qiazol, supplemented with 1 µL of a synthetic RNA mixture containing three different synthetic control RNAs (UniSp2, two fmol/mL; UniSp4, 0.02 fmol/mL; UniSp5, 0.0002 fmol/mL; Qiagen, Hilden, Germany). RNA extraction was performed using 200 µL chloroform, and phase separation was achieved by centrifugation for 15 min at 12,000× g at 4 °C. RNA was extracted from the upper aqueous phase and purified on a QIAcube liquid handling robot using the miRNeasy Mini kit (Qiagen) with the following modifications: glycogen (Ambion, Austin, TX, USA) was added to the aqueous phase to a final concentration of 50 mg/mL and precipitated with 750 µL 100% ethanol. Columns were washed twice with RPE buffer and RNA was eluted in a single round in 30 µL nuclease-free water and stored at –80 °C.
Small RNA Libraries were prepared from 2 µL RNA, using the CleanTag Small RNA Library Prep Kit (TriLink Biotechnologies, San Diego, CA, USA) according to the manufacturer’s protocol. cDNA libraries were amplified in 21 PCR cycles. Equimolar amounts were pooled and libraries underwent 50 cycles of single-end sequencing on a HighSeq 2500 (Illumina, San Diego, USA).

For the preparation of libraries containing 5’-polyphosphate RNAs, total RNA from ES products or gAF worms were treated with 20U 5’ Polyphosphatase (Epicenter) for 30 min followed by ethanol precipitation as recently described [31 ]. For the analysis of small RNA content by next generation sequencing, libraries were prepared using 2.5 μl of total RNA using the CleanTag small RNA Library Prep kit (TriLink), according to the manufacturer’s protocol, using a 1:12 dilution of both adapters. Following initial testing, the final PCR amplification was set at 22 cycles. PCR products of the expected molecular weight (140-160bp) were size selected and samples were sequenced on an Illumina HiSeq high output v4 50bp single-end by Edinburgh Genomics.

Libraries were prepared from 5 μl of miRNeasy RNA using NEXTflex® Small RNA Sequencing Kit v3 for Illumina® Platforms (Bioo Scientific) and QIAseq miRNA Library Kit (QIAGEN) and from 2 μl miRNeasy RNA using CleanTag™ Small RNA Library Prep Kit (TriLink), following each of the manufacturers’ instructions. Additionally, libraries were prepared from 5 μl MagnaZol RNA using NEXTflex® Small RNA Sequencing Kit v3 for Illumina® Platforms (Bioo Scientific) and QIAseq miRNA Library Kit (QIAGEN). Library concentrations were measured using Qubit™ dsDNA HS Assay Kit (Thermo Fisher). Quality and concentration of libraries were determined by Fragment Analyzer (Advanced Analytical). Libraries were sequenced on a NextSeq 500 System (Illumina).

Total microRNA was isolated according to the Total RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada) following the manufacturer’s instructions. microRNA integrity was quantified with the Agilent 2100 Bioanalyzer or 4200 Tapestation (Santa Clara, CA, United States). Libraries were generated utilizing the CleanTag Small RNA Library Prep kit produced by TriLink Biotechnologies (San Diego, CA, United States). In addition, sequencing was conducted on the platform of Illumina HiSeq2000 (San Diego, CA, United States).
Messenger RNA was purified using the Qiagen RNeasy Kit from the adult liver and biliary tree tissue and from isolated cell suspensions of hBTSCs, hHpSCs, hHBs, and hAHeps. RNA integrity analysis was performed using an Agilent 2000 Bioanalyzer. The cDNA libraries were prepared using the Illumina TruSeq Stranded mRNA preparation kit and sequenced on the Illumina HiSeq 2500 platform. More detailed information has also been shown in our previous published work (Oikawa et al., 2015 (link); Dinh et al., 2019 (link)).

CBA × C57BL/6 F1 (CBF1) mice were infected with 400 L3 infective-stage H. bakeri larvae by gavage and adult nematodes were collected from the small intestine 14 days post infection. The nematodes were washed and maintained in serum-free medium in vitro as described previously (17 ). For genomic analyses, DNA was collected from adult worms immediately following harvest from the gut, with extensive washing to remove host material followed by purification using Zymo Research Genomic DNA Clean & Concentrator kit following manufacturer’s instructions (further details in Supplementary Methods). RNA from H. bakeri was collected from adult worms in Qiazol (Qiagen) using mechanical disruption with 5 mm stainless steel beads (Qiagen) on a Tissue Lyser II (Qiagen). Caenorhabditis elegans were harvested and flash frozen as in (18 (link)). Total RNA was treated with RNA 5′ Polyphosphatase (Epicenter) following manufacturer’s instructions, before library preparation. Libraries for small RNA sequencing were prepared using the CleanTag small RNA library prep kit (Trilink) according to manufacturer’s instructions.