GSM6202186: FLT4_TOF-PTV_rep1; Homo sapiens; RNA-Seq
GSM6202186
RNA was harvested using the RNeasy Plus Kit (Qiagen) following manufacturer's instructions. 1 μg of total RNA/sample was used for the generation of sequencing libraries Quality and integrity of the RNA samples were assessed using a 4200 TapeStation (Agilent Technologies) and then libraries generated using the Illumina® Stranded mRNA Prep. Ligation kit (Illumina, Inc.) according to the manufacturer's protocol. Briefly, total RNA (typically 0.025-1ug) was used as input material from which polyadenylated mRNA was purified using poly-T, oligo-attached, magnetic beads. Next, the mRNA was fragmented under elevated temperature and then reverse transcribed into first strand cDNA using random hexamer primers and in the presence of Actinomycin D (thus improving strand specificity whilst mitigating spurious DNA-dependent synthesis). Following removal of the template RNA, second strand cDNA was then synthesized to yield blunt-ended, double-stranded cDNA fragments. Strand specificity was maintained by the incorporation of deoxyuridine triphosphate (dUTP) in place of dTTP to quench the second strand during subsequent amplification. Following a single adenine (A) base addition, adapters with a corresponding, complementary thymine (T) overhang were ligated to the cDNA fragments. Pre-index anchors were then ligated to the ends of the double-stranded cDNA fragments to prepare them for dual indexing. A subsequent PCR amplification step was then used to add the index adapter sequences to create the final cDNA library.