Agilent bioanalyzer platform

RNA was extracted from frozen archival material from cases for which it was available. Prior to extraction, haematoxylin and eosin-stained cryosections were prepared to assess tumour content. Tumour content was graded 1–4 as follows: 1—no evidence of tumour; 2—focal areas of tumour; 3—majority is tumour but with some non-neoplastic components; and 4—extensive tumour/tumour comprised entire section. Cases where the cryosection was graded 1–2 were excluded. Extraction of total RNA was carried out using the miRNeasy Mini Kit according to the manufacturer’s instructions. Eluted RNA was subjected to quality control by analysis on a NanoDrop 1000 spectrophotometer and samples with a 260/280 ratio lower than 1.8 were excluded. Further quality control was carried out using the Agilent Bioanalyzer platform and samples with an RNA Integrity Number (RIN) lower than 4 were excluded. Library preparation and RNA sequencing were carried out by UCL Genomics. Libraries were prepared using non-strand specific Illumina TruSeq Sample Preparation Kits. Libraries were sequenced at a depth of 15 million reads per sample on the Illumina NextSeq 500 platform. Resultant FASTQ files were aligned using TopHat and Cufflinks software packages to produce Binary Alignment Map (BAM) files for subsequent analysis.

For all sequencing experiments, total RNA was isolated from human dermal fibroblasts, as described. The quality of the RNA and prepared libraries was assessed using the appropriate high-sensitivity RNA and DNA microfluidics chip on the Agilent Bioanalyzer platform (Agilent Technologies). All cDNA libraries were subjected to a cleanup step with the use of Agencourt AMPure XP beads (Beckman Coulter) and further multiplexed for sequencing on a HiSeq platform (Illumina).