title: NK cells: Hodgkins Lymphoma,  male, 4 years
design: TruSeq RNA Library Preparation Kit v2 (Cat. # RS-122-2001/RS-122-2002, Illumina Inc.) protocol was used to prepare libraries for total RNA sequencing. First, the ribosomal RNA (rRNA) which constitutes ~95% of the total RNA population were removed using biotinylated, target-specific oligos combined with Ribocop rRNA removal beads (Cat. #037, Lexogen GmBh). Following purification, the ribodepleted RNA was fragmented using divalent cations under elevated temperatures. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase. Second strand cDNA synthesis was performed, using second strand master mix from the kit. The cDNA fragments were then subjected to a series of enzymatic steps which repair the ends, tails the 3 end with a single A base, followed by ligation of the adapters. The adapter ligated products were then purified and enriched using the following thermal conditions: initial denaturation 98C for 30sec; 15 cycles of - 98C for 10sec, 60C for 30sec;72C for 30sec final extension of 72C for 5 mins (TruSeq RNA Library Preparation Kit v2 protocol Cat. #15026495, Illumina Inc.). PCR products were then purified and checked for fragment size distribution on Tape Station using D1000 DNA Screen Tapes (Cat # 5067-5582, Agilent).