Ampure xp reagent

12.5 ng of total DNA were amplified using universal primers targeting the V4 region of the bacterial 16S rRNA gene [64 (link),65 (link)]. Primer sequences contained overhang adapters appended to the 5’ end of each primer for compatibility with Illumina sequencing platform. Master mixes contained 12.5 ng of total DNA, 0.2 μM of each primer and 2x KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, MA). Each 16S rRNA amplicon was purified using the AMPure XP reagent (Beckman Coulter, Indianapolis, IN). In the next step each sample was amplified using a limited cycle PCR program, adding Illumina sequencing adapters and dual‐index barcodes (index 1(i7) and index 2(i5)) (Illumina, San Diego, CA) to the amplicon target. The final libraries were again purified using the AMPure XP reagent (Beckman Coulter), quantified and normalized prior to pooling. The DNA library pool was then denatured with NaOH, diluted with hybridization buffer and heat denatured before loading on the MiSeq reagent cartridge (Illumina) and on the MiSeq instrument (Illumina). Automated cluster generation and paired–end sequencing with dual reads were performed according to the manufacturer’s instructions.

cDNAs were synthesized using a SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific) and used for NGS library preparation. The libraries were manually constructed using our custom thyroid fusion panel, ThyChase. The amplicon library was prepared with the Ion Plus Fragment Library Kit (Life Technologies, Waltham, MA, USA) and the Ion Xpress Barcode Adapters Kit (Life Technologies) according to the manufacturer's instructions. In detail, 10 μL of cDNA was amplified in reaction mixtures of 59 μL containing 45 μL of Platinum PCR SuperMix High Fidelity and 4 μL of ThyChase panel. Polymerase chain reaction (PCR) was performed with a GeneAmp 9700 thermal cycler (Thermo Fisher Scientific) under the conditions of 95°C for 2 min followed by 35 cycles of 95°C for 15 s, 58°C for 15 s, 68°C for 10 s, and a final hold at 4°C. Libraries were purified using 106 μL of AMPure XP Reagent (Beckman Coulter, Miami, FL, USA) on a magnetic stand (Thermo Fisher Scientific) and eluted with 25 μL of low tris-EDTA buffer. Then, adapter ligation and nick repairing were performed to make barcode sequencing adapters (Ion Xpress Barcode Adapters, Thermo Fisher Scientific). Finally, the libraries were quantified using quantitative PCR (qPCR; Ion Library Quantitation Kit, Thermo Fisher Scientific) on a QuantStudio 12K Flex Real-Time PCR System qPCR machine (Thermo Fisher Scientific).

In order to reduce large amplicons containing canonical exon-exon junctions (>150 bp), and to purify the remaining shorter amplicons, all mPCR pools were subjected to a two-step size selection using AMPure XP reagent (Beckman Coulter). In the first step a 1.8x reagent concentration, and in the second step a 3x reagent concentration was used. The size selection process resulted in the purification and enrichment of short 80–150 bp amplicons in the mPCR pools, while the number of large amplicons was reduced (Fig. 3D,E).

Whole transcriptome sequencing was performed for all 23 patients using RNA-Sequencing with Ion AmpliSeq Whole Transcriptome human gene expression kit (Thermo Fisher Scientific, Massachusetts, USA). Briefly, the purified RNAs were evaluated and quantified. Spectrophotometry was performed on the samples and the ratio of absorbance at 260 nm and 280 nm (A260/A280) was used to assess the purity of RNA. A ratio of around 2.0 indicated pure RNA without protein or organic contamination. cDNA library was constructed using Turbo DNase treated RNA using SuperScipt VILO cDNA synthesis kit (Invitrogen, Life Technologies, CA, USA). Samples were then tagged with unique Ion express barcodes and purified using AMPure XP Reagent (Beckman Coulter, USA). Barcoded libraries were assessed using the Ion Taqman library quantitation kit (Thermo Fisher Scientific) and then pooled equally. The pooled libraries were amplified using emulsion PCR on Ion One Touch2 instruments (OT2) and enriched using Ion One Touch ES as per the manufacturer’s instructions. The constructed cDNA libraries were sequenced using an Ion 540 Chip on an Ion S5 XL Semiconductor sequencer (Life Technologies).

Using the primers WP20F1 and WP20R1, the PCR product of the SELEX protocol was purified using a MinElute PCR purification kit (Qiagen). The library was sequenced using the AB library builder system (Thermo Fisher Scientific) and amplified according to the protocol for Ion Xpress Plus and Ion Plus library preparation for the AB library builder system. The library was then purified using the Agencourt AMPure XP reagent (Beckman Coulter). The library size and its concentration were assessed using a Bioanalyzer high-sensitivity chip (Agilent Technologies). The samples were pooled, followed by template preparation on the Ion Chef system, using the Ion PI Hi-Q Chef kit (Thermo Fisher Scientific). The samples were then loaded onto Ion PITM v3 chips and sequenced using the Ion Proton system, with the Ion PITM Hi-Q sequencing 200 kit chemistry (Thermo Fisher Scientific).