RNA-Seq analysis of RT sample TARGET-52-PAWFWK-01A-01R
RNA-Seq analysis of rhabdoid tumor sample TARGET-52-PAWFWK-01A-01R using Illumina HiSeq 2500
Total RNA samples were checked using an Agilent Bioanalyzer RNA nanochip and samples passing quality control were arrayed into a 96-well plate. Polyadenylated (polyA+) RNA was purified using the 96-well MultiMACS mRNA isolation kit on the MultiMACS 96 separator (Miltenyi Biotec, Germany) from 5ug total RNA with on-column DNaseI-treatment as per the manufacturer's instructions. The eluted PolyA+ RNA was ethanol precipitated and resuspended in 10µL of DEPC treated water with 1:20 SuperaseIN (Life Technologies, USA). First-strand cDNA was synthesized from the purified polyA+ messenger RNA using the Maxima H Minus First Strand cDNA Synthesis kit (Thermo-Fisher, USA) and random hexamer primers at a concentration of 5µM along with a final concentration of 1µg/uL Actinomycin D, followed by Ampure XP SPRI bead purification on a Biomek FX robot (Beckman-Coulter, USA). The second strand cDNA was synthesized following the Superscript cDNA Synthesis protocol by replacing the dTTP with dUTP in dNTP mix, allowing second strand to be digested using UNG (Uracil-N-Glycosylase, Life Technologies, USA) in the post-adapter ligation reaction and thus achieving strand specificity (Parkhomchuck et al. 2009). The cDNA was quantified in a 96-well format using a LabChip GX (Perkin Elmer). The cDNA was fragmented by E210 sonication (Covaris) for 55 seconds at a “Duty cycle” of 20% and “Intensity” of 5. The paired-end sequencing library was prepared following the BC Cancer Agency Genome Sciences Centre strand-specific, plate-based and paired-end library construction protocol on a Biomek FX robot (Beckman-Coulter, USA). Briefly, the cDNA was purified in 96-well format using Ampure XP SPRI beads, and was subject to end-repair, and phosphorylation by T4 DNA polymerase, Klenow DNA Polymerase, and T4 polynucleotide kinase respectively in a single reaction, followed by cleanup using Ampure XP SPRI beads and 3’ A-tailing by Klenow fragment (3’ to 5’ exo minus). After purification using Ampure XP SPRI beads, 1uM of Illumina PE adapters were ligated to the cDNA fragments. The adapter-ligated products were purified using Ampure XP SPRI beads, and digested with UNG (1U/µL) at 37oC for 30 min followed by deactivation at 95oC for 15 min. The digested cDNA was purified using Ampure XP SPRI beads, and then PCR-amplified with Phusion DNA Polymerase (Thermo Fisher Scientific Inc. USA) using Illumina’s PE primer set,  with cycle condition 98˚C  30sec followed by 13 cycles of 98˚C  10 sec, 65˚C  30 sec and 72˚C  30 sec, and then 72˚C  5min. The PCR products were purified using Ampure XP SPRI beads, and checked with LabChip GX for DNA samples using the High Sensitivity Assay (PerkinElmer, Inc. USA). PCR product of the desired size range was bead purified, and the library profile assessed and quantified using an Agilent DNA 1000 series II assay and Quant-iT dsDNA HS Assay Kit using Qubit fluorometer (Invitrogen), then diluted to 8nM and pooled. The pooled library concentration was determined by Quant-iT dsDNA HS Assay prior to Illumina Sequencing.
BCCAGSC
A37344-1