ArrayExpress

Clustered on cBot and sequenced on Illumina HiSeq 2000/2500 according to manufacturers instructions.

Illumina Truseq RNA v2

The use of human tissue samples was approved by the Uppsala Ethical Review Board (Reference #2011/473). Tissues samples, collected within the infrastructure of an established biobank, were embedded in Optimal Cutting Temperature (O.C.T.) compound and stored at -80C. A hematoxylin-eosin (HE) stained frozen section (4um) was prepared from each sample using a cryostat and the CryoJane Tape-Transfer System (Instrumedics, St. Louis, MO, USA). Each slide was examined by a pathologist to ensure proper tissue morphology. Three sections (10um) were cut from each frozen tissue block and collected into a tube for subsequent RNA extraction.

The tissue was homogenized mechanically using a 3 mm steel grinding ball (VWR). Total RNA was extracted from cell lines and tissue samples using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers instructions. The extracted RNA samples were analyzed using either an Experion automated electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA) with the standard-sensitivity RNA chip or an Agilent 2100 Bioanalyzer system(Agilent Biotechnologies, Palo Alto, USA) with the RNA 6000 Nano Labchip Kit. Only samples of high-quality RNA (RNA Integrity Number 7.5) were used in the following mRNA sample preparation for sequencing.

2x100 bp paired end sequencing

Libraries were constructed using the TruSeq v 2 kit, with poly-A-enrichment or with the TruSeq Stranded kit (see library strand annotation).

The tissue was homogenized mechanically using a 3 mm steel grinding ball (VWR). Total RNA was extracted from cell lines and tissue samples using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers instructions. The extracted RNA samples were analyzed using either an Experion automated electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA) with the standard-sensitivity RNA chip or an Agilent 2100 Bioanalyzer system (Agilent Biotechnologies, Palo Alto, USA) with the RNA 6000 Nano Labchip Kit. Only samples of high-quality RNA (RNA Integrity Number 7.5) were used in the following mRNA sample preparation for sequencing.