title: RNAseq of primary endemic BL tumor: Sample R0022
design: "Strand Specific RNAseq Library Preparation. Starting with 1-5ug total RNA, we isolated mRNA with oligo-dT using Dynabeads mRNA purification kit (Life Technologies). mRNA was fragmented with metal ions and after the first strand synthesis with Superscript III, we marked the second strand by incorporating dUTP instead of dTTP. Then end repair and A-tailing steps were followed by Y-shaped Illumina adapter ligation step which maintains strand directionality. We size selected libraries using XP Ampure magnetic beads (Beckman Coulter Inc.) selecting for a 330 bp insert size. Prior to low cycle PCR with High Fidelity Phusion polymerase (NEB) for amplification and adapter sequence completion, second strand marked with dUTP was degraded. During the sequencing library preparation we checked DNA quality and fragment size distribution using Bioanalyzer Agilent 1000 kit. Final library qualities were confirmed with Bioanalyzer Agilent High sensitivity DNA kit and sequenced with paired end read (2x100bp) using an Illumina HiSeq 2000 (Illumina Inc., San Diego, CA, USA)."