title: GSM1527983: N121_MB_24H; Bos taurus; RNA-Seq
library_construction_protocol: 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.
attributes.tag: GEO Accession
attributes.value: GSM1527983