Bioanalyser rna nanochip

Total RNA was extracted from the abomasal biopsies as follows: the 645 abomasal biopsy samples were homogenised in RLT buffer (Qiagen Ltd, UK) using a Precellys bead basher (Bertin Instruments, UK) with CK28 bead tubes (Stretton Scientific, UK). Samples were centrifuged at 14,000 × g at 4 °C for 10 min and the supernatant collected for processing using a RNeasy mini-isolation kit (Qiagen Ltd, UK) according to the manufacturers’ protocol, including an on-column DNase digestion. RNA quality and integrity were assessed using a Nanodrop spectrophotometer (Thermo Fisher, UK) and a Bioanalyser RNA Nanochip (Agilent Technologies Ltd, UK). The yield of total RNA was determined on a Qubit Fluorometer (Thermo Fisher, UK) using the Broad Range RNA kit (Thermo Fisher, UK). The RNA isolated from three biopsies per animal for each time-point was pooled 1:1:1 by weight to generate the final samples for RNA-seq assessment. All samples exceeded RIN ≥ 8.2. The resulting 180 RNA samples were sequenced on an Illumina NextSeq 500 by Glasgow Polyomics⁷⁰ generating 75 bp paired-end reads at an average sequencing depth of 25 Mbp/sample. FastQC analysis indicated that all sequences exceeded Phred Scores of 30 along all 75 bp.

250 μl of culture were harvested from late exponential and stationary phase by centrifugation (15,000 x g at 4°C for 15 min). The supernatant was discarded and the pellet re-suspended in 100 μl of Y1 Buffer (1M Sorbitol, 0.1M EDTA, 1 mg/ml lysozyme, 0.1% β-mercaptoethanol) and incubated at 37°C for 1 hr at 500 rpm. The cell suspension was added to 350 μl of RLT buffer, 250 μl 100% ethanol and loaded onto an RNeasy column from the RNeasy Kit (Qiagen, Germany). RNA was then washed and eluted following the manufacturer’s protocol. Eluted samples were incubated with DNase I (New England Biolabs, MA) for 10 min at 37°C and then cleaned up with a second passage through the RNeasy column (loading, washes and elution according to manufacturer’s instructions). Samples were finally eluted in 30 μl of RNase-free water and RNA quantified with Nanodrop. Quality assessment of the extracted RNA was carried out with an Agilent Bioanalyser RNAnano chip and five replicates per strain/condition were chosen for sequencing.

Post stimulation with IL-4 and anti-CD40, cells were harvested at 0, 12, 36, 72, 120 and 288 hours. RNA was extracted using the RNeasy MINI Kit (Qiagen Ltd.). Quantity and quality of RNA was assessed using a spectrophotometer and Bioanalyser RNA Nano Chip (Agilent Technologies Ltd) respectively. One μg of high quality RNA was synthesised to double-stranded cDNA using the one-cycle cDNA kit (Affymetrix Ltd.) after RNA reduction to reduce 18 S RNA. In vitro transcription of cRNA was carried out using the IVT kit (Affymetrix Ltd.) resulting in complementary amplification and biotin labelling. cRNA was purified, concentrated and checked for quality and quantity. Ten μg of cRNA was fragmented and hybridised onto a Human Exon 1.0 ST Array (Affymetrix Ltd) for 16 hours. GeneChips were washed, stained on a Fluidics 450 station and scanned using a high-resolution scanner (Affymetrix Ltd.).