Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Two RNA-seq studies, one of coding and the other of coding and non-coding RNA for Identification of meningococcal factors required for colonization of epithelial and endothelial cells by high throughput screening

ABSTRACT: RNA-Seq analyses were performed in N. meningitidis strain Z5463 by deep sequencing using Ion Torrent technology (Thermo Fischer Scientific). For whole transcriptome libraries (mRNA+sRNA), cDNA libraries were prepared using the Ion Total RNA-Seq Kit v2 (Life Technologies) including a prior step of ribodepletion using specific probes adapted to N. meningitidis (Low Input Ribo Minus Eukaryote System v2) and size fractionation of RNA prior to cDNA synthesis with RNase III was performed. Briefly, 500 ng of total RNA were ribodepleted and then fragmented. After fragmentation, mean size of fragment was around 120 nt. Coding_and_NonCoding_R1, Coding_and_NonCoding_R2, and Coding_and_NonCoding_R3 represent three technical replicates. For sRNA libraries (sRNA), three cDNA libraries were prepared using the Ion Total RNA-Seq Kit v2 for Small RNA Libraries (Life Technologies) including a prior step of enrichment in small RNA fraction. Equal amounts of total RNA were used for the generation of all cDNA libraries. The three whole transcriptome libraries libraries (mRNA+sRNA) were then sequenced on an Ion ProtonTMSystem using the Ion PI Template and Sequencing OT 200 kit v3 and the 3 sRNA libraries were sequenced on an Ion PGM with the Hi-Q Template and Sequencing Kit using a 316 v2 array. NonCoding_R1, NonCoding_R2, and NonCoding_R3 represent three technical replicates.

INSTRUMENT(S): Ion Torrent Proton

ORGANISM(S): Neisseria meningitidis

SUBMITTER: Thomas Nussbaumer

PROVIDER: E-MTAB-4768 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

Dataset Information

Two RNA-seq studies, one of coding and the other of coding and non-coding RNA for Identification of meningococcal factors required for colonization of epithelial and endothelial cells by high throughput screening

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