title: RNA-seq of human sarcoma
design: RNA extraction from frozen samples was performed using a standard TRIzol (Life Technologies) / chloroform extraction followed by 70% ethanol precipitation and RNA purification using the RNeasy Mini Kit (Qiagen) with a DNase treatment (RNase-Free DNase Set, Qiagen). RNA extraction from FFPE tissue was performed using a Deparaffinization Solution (Qiagen) as recommended by the manufacturer followed by a DNase treatment (RNase-Free DNase Set, Qiagen) and RNA purification using the RNeasy FFPE kit (Qiagen) according to the manufacturers recommendations. RNA were quantified using a Nanodrop 1000 spectrophotometer (Thermo Scientific) and were qualified with the Agilent 2100 Bioanalyzer (Agilent) using the Agilent RNA 6000 Nano Kit (Agilent) according to the manufacturers instructions. An ERCC RNA Spike-In Mix (Life technologies) was added to each RNA as recommended by the manufacturer.The RNA-seq libraries from FFPE tissue samples total RNA were prepared using a modified TruSeq RNA Sample Prep Kit v2 protocol. Briefly, rRNA was depleted from 0.5 g of total RNA using the RiboZero Magnetic Gold Kit (Human/Mouse/Rat, Epicentre). Ribosomal RNA-depleted RNA samples were purified using Agencourt RNA Clean XP beads (Beckman Coulter Genomics) and RNA was eluted with the Elute, Prime, Fragment Mix from the TruSeq RNA Sample Prep Kit v2 (Illumina Inc.). The RNA fragmentation time (2.5 to 5 minutes) varied depending on the quality of the initial total RNA (assessed by Eukaryote Total RNA Nano Bioanalyzer assay, Agilent). Following the fragmentation, first and second strand synthesis, the Illumina barcoded adapters were ligated at 1/10 dilution of the recommended concentration. Libraries were enriched with 15 cycles of PCR. The size and quality of the libraries were assessed in a High Sensitivity DNA Bioanalyzer assay (Agilent). Starting input material for the libraries construction was DNA free total RNA from frozen tissue using the TruSeq RNA Sample Prep Kit v2 (Illumina Inc.,) according to manufacturers protocol. In short, 0.5 g of total RNA was used for poly-A based mRNA enrichment selection followed by fragmentation by divalent cations resulting into fragments of 80-250nt, with the major peak at 130nt. Double stranded cDNA was end repaired, 3adenylated and the 3-T nucleotide of barcoded Illumina adapters was used for the adapter ligation. The ligation product was amplified with 12 cycles of PCR and quality controlled in Agilent DNA 7500 Bioanalyzer assay (Agilent). Each library was sequenced using TruSeq SBS Kit v3-HS, in paired end mode with the read length 2x76bp. We generated minimally 23 million paired end reads passing filter for each FFPE RNA-seq library or at least 35 million paired end reads passing filter for each frozen tissue RNA-seq library in a fraction of a sequencing lane on HiSeq2000 (Illumina, Inc) following the manufacturers protocol. Image analysis, base calling and base quality scoring of the run were processed by integrated primary analysis software - Real Time Analysis (RTA 1.13.48) and followed by generation of FASTQ sequence files by CASAVA (v1.8).