5 protocols
high throughput sequence alignment protocol
Bowtie to Hg19 and Mm9
nucleic acid sequencing protocol
Libraries were sequenced on the SOLiD platform (Applied Biosystems). Initially, sequencing of the 10bp library barcode was performed followed by 35bp reverse and 50bp forward paired-end sequencing. Only the 50bp forward reads were used in this study.
nucleic acid extraction protocol
~50mg of tissue were cut from the frozen tumours and RNA isolated using the RNeasy Lipid Tissue Mini Kit (Qiagen) according to manufacturer's instructions. On-column DNase digestion was performed using the RNase-free DNase Kit (Qiagen).
nucleic acid library construction protocol
Total RNA was cleaned using the Ribominus concentration module (Invitrogen) and rRNA depletion was carried out with the Dynabeads mRNA purification kit (Invitrogen). Libraries enriched with polyA mRNA suitable for sequencing by the SOLiD platform were created using the SOLiD Total RNA Seq Kit supplied by Applied Biosystems in combination with the SOLiD RNA Barcoding Kit, Module 1-16. In each instance, RNase III digested polyA RNA was used as input into library creation and 15 cycles of amplification were employed to produce the final libraries.
growth protocol
Cells for tumour implantation were grown in DMEM supplemented with glutamine and 10% foetal calf serum and implanted subcutaneously in nude mice.