Biopsies (~25 mg) were lysed in a 300 µl RNeasy Fibrous Tissue kit (Qiagen, Hilden, Germany) RLT buffer and 0.14 M β-mercaptoethanol (β-ME) (Sigma-Aldrich, St. Louis, USA), and then homogenised by centrifugation at 10,000 g through a Qiashredder column (Qiagen). Protein was removed by incubation for 10 minutes at 55°C with 10 µl Proteinase K (20 mg/ml) (>600 mAU/ml) (Qiagen). Total RNA was extracted on RNeasy Mini spin columns and DNA was removed with RNase free DNase digestion (Qiagen). Samples were included in the microarray experiment if they had an optical density ratio reading of 1.8-2.0 OD260/OD280 and >1.8 OD260/OD230 as well as a RNA concentration of greater than 50 ng/μl. For each biopsy sample, 500 ng of total RNA was amplified and purified using the Illumina TotalPrep-96 RNA Amplification kit (Ambion®, Life Technologies™, CA, USA).
Label
Biotin, Cy3
Label protocol
750 ng of biotin-labelled cRNA (150 ng/μl) was hybridised to Illumina HumanHT-12v4 beadarrays (Illumina, CA, USA) for 16 hours at 58°C.
Hybridization protocol
750 ng of biotin-labelled cRNA (150 ng/μl) was hybridised to Illumina HumanHT-12v4 beadarrays (Illumina, CA, USA) for 16 hours at 58°C.
Scan protocol
Following hybridisation, beadarrays were washed and stained with streptavidin-Cy3 (GE Healthcare, UK), scanned using the Beadarray reader, and processed using Genome Studio software (Illumina).
Data processing
Expression data for Illumina HumanHT-12v4 beadarrays was normalised after Log2 transformation within Genome Studio (Illumina) using cubic spline normalisation followed by ComBat normalisation for chip effects in R.
Mucosal transcriptomics implicates under expression of FAM5C in the pathogenesis of ulcerative colitis
Data table header descriptions
ID_REF
VALUE
cubic spline normalized (Probes that reached a minimum detection p-value of p<0.01 in at least two biopsies in any location were included in the subsequent analyses (n=26,261 probes)
