GSM9292361: RNA-seq, mRNA, untreated, patient 3 after 6 months of imatinib; Homo sapiens; RNA-Seq
Trizol (TRI Reagent) is used to isolate total RNA from whole cells. mRNA is purified from approximately 200ng of total RNA with oligo-dT beads and sheared by incubation at 94C in the presence of Mg (Roche Kapa mRNA Hyper Prep Cat# KR1352). Samples are then fragmented using heat and magnesium. 1st strand synthesis is performed using random priming.  2nd strand synthesis incorporates dUTPs into the 2nd strand cDNA.  Adapters are then ligated and the library is amplified.  Strands marked with dUTPs are not amplified allowing for strand-specific sequencing.  Indexed libraries that meet appropriate cut-offs for both quantity and quality are quantified by qRT-PCR using KAPA Biosystems kit (# KK4854) and insert size distribution determined with Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/ul and a size distribution of 150-300bp are used for sequencing.