GSM9690032: A549 Cells, U2AF1 S34F TTC codon, sample 3, long-read; Homo sapiens; RNA-Seq
RNA for each cell clone was extracted using the RNeasy Plus Mini Kit (QIAGEN, 74134) and quantified using the Qubit RNA Broad Range Quantification Assay Kit (Invitrogen, Q10210) Long-read RNA-sequencing was performed with Broad Clinical Labs. An aliquot of 300ng of total RNA was used as the input for Kinnex™ full length cDNA synthesis and amplification (PacBio Iso-Seq express 2.0 kit, 103-071-500). The amplified cDNA was then quantified by Qubit and Tapestation (Qubit dsDNA HiSens, QUBDSDNA500KT, and HiSense D5000 ScreenTape, 50675592) and barcoded cDNA was then pooled before proceeding to 8-fold Kinnex PCR (PacBio Kinnex PCR 8-fold kit, 103-071-600). Samples then were programmatically concatenated into single molecules optimal for long-read sequencing (Kinnex full-length RNA kit, 103-072-000). Success of array formation was evaluated through qubit quantification and Agilent TapeStation fragment size QC (Qubit dsDNA HiSens, QUBDSDNA500KT, and GenomicDNAScreenTape, 50675366). Each pooled Kinnex library underwent sequencing preparation using the Revio polymerase kit (Pacific Biosciences, 102-739-100). Using the Sample Setup page in SMRTLink, the appropriate volumes of each reagent was calculated by factoring in the insert size, sample concentration, and target plate loading concentration of 150pM. The run design was set up with the Application Type "Kinnex full-length RNA" and Library Type "Kinnex." Samples were loaded onto the sequencer within 24 hours of completed sequencing preparation and were sequenced using the 24-hour movie setting.