All bisulphite-modified DNA samples were amplified using separate multiplex or simplex PCRs, as listed in Supplementary Table 1. The assay designed using Assay Design Service (ADS) software for genes or regions is described in Supplementary Table 2. The PCRs included 0.5 units of Qiagen HotStarTaq, 0.2 µM primers, and 3 µl of bisulphite-treated DNA in a 20 µl reaction. All PCR products were verified and quantified using the QIAxcel Advanced System. Prior to library preparation, PCR products from the same sample were pooled and purified using QIAquick PCR Purification Kit columns (Qiagen). Libraries were prepared using a KAPA Library Preparation Kit for Ion Torrent Platforms (cat# KK8310) and Ion Xpress™ Barcode Adapters (Thermo Fisher). Next, the libraries were purified using Agencourt AMPure XP beads (Beckman Coulter) and quantified using the Qiagen QIAxcel Advanced System. The barcoded samples were pooled in an equimolar fashion before template preparation, and enrichment was performed on an Ion Chef™ system (Thermo Fisher) using Ion 520™ & Ion 530™ Chef reagents. Next, the enriched, template-positive libraries were sequenced on an Ion S5™ sequencer using Ion 530™ sequencing chips (Thermo Fisher).
Ion 530 sequencing chips
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ion 530™ sequencing chips are designed for use with the Ion Torrent™ next-generation sequencing platform. They provide a high-throughput solution for DNA or RNA sequencing applications.
Automatically generated - may contain errors
Lab products found in correlation
Ion chef system, by Thermo Fisher Scientific (4 mentions)
Kapa library preparation kit for ion torrent platforms, by Thermo Fisher Scientific (4 mentions)
Agencourt ampure xp bead, by Beckman Coulter (4 mentions)
Ion xpress barcode adapter, by Thermo Fisher Scientific (4 mentions)
Qiaxcel advanced system, by Qiagen (4 mentions)
Hotstartaq, by Qiagen (4 mentions)
Qiaquick pcr purification kit columns, by Qiagen (2 mentions)
Ez 96 dna methylation kit, by Zymo Research (2 mentions)
4 protocols using ion 530 sequencing chips
1
Targeted Bisulfite Sequencing Protocol
2
Ion Torrent-Based DNA Methylation Analysis
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All bisulfite-modified DNA samples were amplified using separate multiplex or simplex PCRs. The assay designed using Assay Design Service (ADS) software (v1.0.6, QIAGEN, Hilden, Germany) for genes or regions. The PCRs included 0.5 units of Qiagen HotStarTaq, 0.2 µM primers, and 3 µL of bisulfite-treated DNA in a 20 µL reaction mixture. QIAxcel Advanced System (v1.3.0, QIAGEN, Hilden, Germany) were used to quantify the PCR products. Meanwhile, PCR products from the same sample were pooled and purified using QIAquick PCR Purification Kit columns (Qiagen). Libraries were prepared using a KAPA Library Preparation Kit for Ion Torrent Platforms (cat# KK8310) and Ion Xpress™ Barcode Adapters (Thermo Fisher, Waltham, MA, USA). Next, the libraries were purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and quantified using the Qiagen QIAxcel Advanced System. The barcoded samples were pooled in an equimolar fashion before template preparation, and enrichment was performed on an Ion Chef™ system (Thermo Fisher, Waltham, MA, USA) using Ion 520™ and Ion 530™ Chef reagents. Next, the enriched, template-positive libraries were sequenced on an Ion S5™ sequencer using Ion 530™ sequencing chips (Thermo Fisher, Waltham, MA, USA).
3
Targeted Bisulfite Sequencing of BAL DNA
4
Targeted Bisulfite Sequencing of BAL DNA
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Targeted next-generation bisulfite sequencing (tNGBS) was performed on nine of the original 13 BAL specimens measured in the DNA EPICmethylation array by EpigenDx (Hopkinton, MA). DNA bisulfite modification was done using EZ-96 DNA methylation kit (Zymo), followed by multiplex PCR with Qiagen HotStar Taq and products purified with QIAquick PCR purification kit. Libraries were prepared using the KAPA Library Preparation Kit for Ion Torrent platforms and Ion Xpress Barcode Adapters (Thermo Fisher Scientific). Library products were purified using Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN) and quantified using the Qiagen QIAxcel Advanced System. Barcoded samples were then pooled in an equimolar fashion before template preparation and enrichment were performed on the Ion Chef system (Thermo Fisher Scientific) using Ion 520 and Ion 530 Chef reagents. Enriched, template-positive library products were sequenced on the Ion S5 sequencer using Ion 530 sequencing chips (Thermo Fisher Scientific). FASTQ files from the Ion Torrent S5 server were aligned to the local reference database using open-source Bismark Bisulfite Read Mapper with the Bowtie2 alignment algorithm. Methylation levels were calculated in Bismark by dividing the number of methylated reads by the total number of reads.
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