GSM6339464
RNA was extracted with TRI Reagent (Sigma-Aldrich) from the BT-474, MCF7, and T47D cell lines (purchased from ATCC) according to the manufacturer's instructions and treated with DNase I (Thermo Fisher Scientific). The FirstChoice Human Total RNA Survey Panel with normal tissue RNA was purchased from Ambion/Thermo Fisher Scientific. First-strand cDNA was generated using RevertAid H minus (Thermo Fisher Scientific) and an anchored oligo(dT) primer (Integrated DNA Technologies) as per the manufacturer's recommendations. Primers for reverse transcription polymerase chain reaction (RT-PCR) were designed to amplify full-length transcripts from seven different locations in first exons. PCR was done using AccuPrime GC-Rich DNA Polymerase (Thermo Fisher Scientific) with the following conditions: 98 °C for 3 minutes, followed by 34 cycles of 95 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for 2 minutes, and a final extension step of 72 °C for 10 minutes. Products were purified using AMPure XP beads (Beckman Coulter) and quantitated with the Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific) before pooling all amplicons per sample at equimolar concentrations to a final mass of 150 ng (~140 fmol) per pool. Pools underwent end-repair with the NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs) before clean-up with AMPure XP beads. Barcoding ligation was performed with the PCR Native Barcoding Expansion 1-12 PCR-free kit (Oxford Nanopore Technologies) and Blunt/TA Ligase Master Mix (New England Biolabs) to allow sample pooling and multiplexing. Barcoded amplicon pools were purified using AMPure XP beads and quantitated with the Qubit dsDNA High Sensitivity Assay Kit before pooling equimolar amounts of each barcoded sample (1-12) to reach a final concentration of 200 femtomoles. Library quality was assessed on a Bioanalyzer with a High Sensitivity DNA chip (Agilent Technologies) and measured using the Qubit dsDNA Broad Range Assay kit. Adapter ligation was performed using the Ligation Sequencing Kit (Oxford Nanopore Technologies), followed by purification using AMPure XP beads and elution in Elution Buffer.