Project description:The ligation step in RNA sequencing library generation is a known source of bias. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig). We also investigate whether using the thermostable ligase Methanobacterium thermoautotrophicum RNA ligase K97A (Mth K97A) for the initial ligation step in the CircLigase protocol reduces bias. A pool of small RNA fragments of known composition was converted into a sequencing library using one of three protocols and sequenced on an Ion Torrent PGM. The single adaptor CircLigase-based approach significantly reduces, but does not eliminate, bias in Ion Torrent data. Using Mth K97A as part of the CircLig method does not further reduce bias.
Project description:Next generation sequencing using the Ion Torrent PGM platform after PNLDC1 silencing in mouse embryonic stem cells E14 using esiRNA
2016-09-12 | GSE83405 | GEO
Project description:Ion Torrent PGM sequencing for microbial ecology
Project description:Exosomes/microvesicles (hereafter referred to as extracellular vesicles) were isolated from the ULF of day 14 cyclic and pregnant ewes using ExoQuick-TC. Extracellular vesicle RNA was pooled (n=4 per status) and analyzed for small RNAs by sequencing on the Ion Torrent PGM platform and analysis with CLC Genomics Workbench small RNA workflow based on the miRBase (Release 19) Bos taurus database.
Project description:Purpose: to reveal the small RNA profile of postnatal retina in Wistar WU albino rat by Ion-Torrent PGM sequencing technology. Methods : Wistar WU rats ranging in age from P1 to P21 were anesthetized by inhalation using Forane prior to sacrifice at the same hour to avoid circadian variation. Total RNA was extracted from the retinal tissues of various developmental stages using NucleoSpin miRNA kit (Macherey–Nagel, Düren, Germany) following manufacturer's instructions. The miRNA concentration was assessed using Qubit microRNA Assay Kit (ThermoFisher Scientific, MA, USA). The RNA integrity number (RIN), an algorithm for judging the integrity of RNA samples, were evaluated using Agilent 2100 Bioanalyzer (Agilent Technologies, USA) following the manufacturing instruction of the RNA 6000 Nano kit and RIN>7 was considered acceptable. MicroRNAs was also determined using the Agilent Small RNA Kit to have deeper view in the 10 to 40-nucleotide size range. Small RNA library construction from pooled retinal samples (N=3, in each age-group) were carried out according to the Ion Total RNA-Seq Kit v2 protocol (Revision E) with slight modification. Enzymatic reaction was performed in half reaction volume, while cleaning procedures were executed with the accurate final volume according to the protocol. The Ion 316 or 318 ™ Chip v2 was used for sequencing on the Ion Torrent PGM™ instrument according to the protocol of Ion PGM™ Hi-Q View Sequencing Kit. To assess reliability of miRNA workflow solution in 316 v2 chip, two independent P7 samples were assayed (biological replicates) and P21 sample were sequenced as technical replicates in every sequencing procedure. Some samples were also sequenced in 318 v2 chip (P3, P5, P10, P15 and P21) as biological replicate. Ion Torrent Suite Platform was used to trim the raw sequence data and remove any residual sequencing adapter fragments that remained on the 5′ or 3′ ends. Reads were mapped to the non-coding RNAs from ENSEMBL [Rnor_6.0 (GCA_000001895.4)] using TMAP algorithm. These aligned BAM (Binary Alignment Map) files were further processed in Galaxy Web-based platform (Afgan, 2018) via Cufflinks, Cuffmerge and Cuffdiff (Version 2.2.1.3) application (Trapnell, 2010). Further analysis and visualising of the datasets was carried out in R Studio Software environment. qRT–PCR validation was performed using TaqMan assays.
Project description:To understand the effect of Babesia infection on Rhipicephalus microplus hemocyte gene expression, we performed high throughput RNA-sequencing using samples collected from Rhipicephalus microplus uninfected tick hemolymph and infected with either Babesia bigemina or Babesia bovis. We evaluated gene expression differences that may be attributed to tick immune defense to babesial infection.
2023-12-31 | GSE243493 | GEO
Project description:Bacterial diversity of the tick Rhipicephalus sanguineus