Illumina HiSeq 2000 paired end sequencing; GSM1527971: N1178_CN_24H; Bos taurus; RNA-Seq

N1178_CN_24H

Mycobacterium bovis, the agent of bovine tuberculosis, causes an estimated $3 billion annual losses to global agriculture due, in part, to the limitations of current diagnostics. Development of next-generation diagnostics requires a greater understanding of the interaction between the pathogen and the bovine host. Therefore, to explore the early response of the alveolar macrophage to infection, we report the first application of RNA-sequencing to define, in exquisite detail, the transcriptomes of M. bovis-infected and non-infected alveolar macrophages from ten calves at 2, 6, 24 and 48?hours post-infection. Differentially expressed sense genes were detected at these time points that revealed enrichment of innate immune signalling functions, and transcriptional suppression of host defence mechanisms (e.g., lysosome maturation). We also detected differentially expressed natural antisense transcripts, which may play a role in subverting innate immune mechanisms following infection. Furthermore, we report differential expression of novel bovine genes, some of which have immune-related functions based on orthology with human proteins. This is the first in-depth transcriptomics investigation of the alveolar macrophage response to the early stages of M. bovis infection and reveals complex patterns of gene expression and regulation that underlie the immunomodulatory mechanisms used by M. bovis to evade host defence mechanisms. Overall design: Whole-transcriptome analysis of M. bovis- and non-infected alveolar macrophages from ten calves (n = 10) at 2, 6, 24 and 48 hours (h) post-infection using RNA-sequencing (RNA-seq).

RNA sequencing provides exquisite insight into the manipulation of the alveolar macrophage by tubercle bacilli

Submitted by Gene Expression Omnibus on 12-OCT-2015

All RNA extractions were performed using an RNeasy® Plus Mini kit (Qiagen Ltd.) according to the manufacturer’s instructions. RNA quantity and quality was assessed using a NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific Inc.) and an Agilent 2100 Bioanalyzer with a RNA 6000 Nano kit (Agilent Technologies Ltd., Cork, Ireland). All samples displayed a concentration ranging from 10 up to 135 ng/μl (median 50 ng/μl), a 260/280 ratio greater than 2.0 and RNA integrity numbers greater than 8.5. RNA samples were stored at −80 °C until required. For the current study, 78 strand-specific RNA-seq libraries were prepared. These comprised M. bovis- and non-infected samples from each post-infection time points (2, 6, 24 and 48 h) across 10 animals (with the exception of one animal that did not yield sufficient alveolar macrophages for in vitro infection at the 48 h post-infection time point). For RNA-seq library preparation, 200 ng of total RNA from each sample was used to prepare individually barcoded strand-specific RNA-seq libraries. Two rounds of poly(A)+ RNA purification were performed for all RNA samples using the Dynabeads® mRNA DIRECT™ Micro Kit (Invitrogen™, Life Technologies Corp.) according to the manufacturer’s instructions. The purified poly(A)+ RNA was then used to generate strand-specific RNA-seq libraries using the ScriptSeq™ v2 RNA-Seq Library Preparation Kit, the ScriptSeq™ Index PCR Primers (Sets 1 to 4) and the FailSafe™ PCR enzyme system (all sourced from Epicentre®, Illumina® Inc., Madison, WI, USA) according to the manufacturer’s instructions. RNA-seq libraries were purified using the Agencourt® AMPure® XP system (Beckman Coulter Genomics, Danvers, MA, USA) according to the manufacturer’s instructions for double size selection (0.75× followed by 1.0× ratio). RNA-seq libraries were quantified using a Qubit® fluorometer and Qubit® dsDNA HS Assay Kit (Invitrogen™, Life Technologies Corp.), while library quality checks were performed using an Agilent 2100 Bioanalyzer and High Sensitivity DNA Kit (Agilent Technologies Ltd.). Individually barcoded RNA-seq libraries were pooled in equimolar quantities and the quantity and quality of the final pooled libraries (three pools in total) were assessed as described above. Cluster generation and sequencing of the pooled RNA-seq libraries were performed by BGI (BGI–Hong Kong, Hong Kong, China) using an Illumina® HiSeq™ 2000 sequencer. Each of the pooled libraries was sequenced as paired-end 2 × 90 nucleotide reads on eight Illumina® flow cell lanes.

RNA sequencing provides exquisite insight into the manipulation of the alveolar macrophage by tubercle bacilli

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