Ion pi hi q sequencing 200 kit

RNA-seq was conducted as previously described [67 ]. Initially, intact mRNA isolated from total RNA (1000 ng) was purified using the Dynabeads™ mRNA DIRECT™ Micro Purification kit (Cat# 61,021; Ambion). The ribosomal RNA was depleted using the RiboMinus™ Eukaryote System v2 (Cat# A15026; Life Technologies, Carlsbad, Canada). Next, cDNA transcriptome libraries were constructed using the Ion Total RNA Seq kit v2 (Cat# 4,475,936; Life Technologies), incorporating barcodes from the Ion Xpress RNA-Seq Barcode 01–16 kit (Cat# 4,475,485; Life Technologies). The whole-transcriptomic libraries were diluted to 100 pM and amplified using the Ion OneTouch™ 2 System and Ion PI Hi-Q OT2 200 kits (Cat# 4,474,779; Life Technologies). The template-positive ion-sphere particles were sequenced using the Ion PI Hi-Q Sequencing 200 kit (Cat# A26433; Life Technologies). These ion-sphere particles were loaded and incubated with polymerase on a chip from Ion PI Chip kit v3 (Cat# A26771; Life Technologies). RNA-seq was performed using an Ion Proton System (Life Technologies), and all experimental procedures were performed according to the manufacturer's instructions.

Genomic DNA was extracted using a QIAamp DNA blood mini kit (Qiagen, Hilden, Germany) from 400 μl peripheral blood. The DNA qualities and quantities were verified with a Nanodrop spectrometer (Nanodrop Technologies, Wilmington, DE, USA) and a Qubit fluorometer (Life Technologies, Grand Island, NY, USA). Amplicon libraries were prepared according to the manufacturer's instructions using the Ion PI library kit plus an Ion PI Ampliseq Exome RDY 1 × 8 (Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA, USA), and an Ion PI Hi‐Q OT2 200 kit was used for template preparation according to the manufacturer's instructions. Sequencing was conducted with an Ion PI Hi‐Q Sequencing 200 kit and an Ion PI Chip v3 in a Proton Semiconductor Sequencer (Life Technologies, Thermo Fisher Scientific Inc.).

We loaded the enriched ISPs onto Ion PI Chip v3, using 30 samples on a chip per sequencing run with the Ion PI HIQ Sequencing 200 kit (Life Technologies). For sequencing, we used 520 flow runs that generated approximately 200-bp reads. Ion Torrent sequencing was performed using the Ion PI HIQ Sequencing 200 kit, following the standard protocol.

cDNA libraries for single-end sequencing were prepared using the Ion Total RNA-seq kit, v2.0 (Life Technologies). Samples were diluted and mixed, and the mixture was processed using a OneTouch 2 instrument (Life Technologies) and enriched on a OneTouch 2 ES station (Life Technologies) to prepare template-positive Ion PI ion sphere particles (Life Technologies) using the Ion PI Hi-Q OT2 200 kit (Life Technologies). After enrichment, the mixed template-positive Ion PI ion sphere particles for each sample were loaded onto a P1v3 Proton Chip (Life Technologies) and sequenced on an Ion Proton sequencer using the Ion PI Hi-Q sequencing 200 kit (Life Technologies). The DESeq algorithm was used to identify differentially expressed genes. Gene Ontology (GO) analysis was performed to elucidate the biological implications of the differentially expressed genes. We also validated the expression of changed genes in SW480 and LoVo cells following LINC01296 downregulation by qRT-PCR.