Long-read ONT-dRNA and PacBio Kinnex full-length RNA sequencing of UPF1 depletion in human colorectal adenocarcinoma cell line HCT116 via the auxin-inducible degron (AID) system
Cells were harvested and lysed by adding 2 ml Invitrogen™ TRIzol™ Reagent (Invitrogen; Cat# 15596018) to each plate. The N-AID-UPF1-tagged HCT116 cell line was maintained at 37°C and 5% CO2 in a humidified incubator in McCoy's 5A (Modified) Medium (Gibco), supplemented with 10% fetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco). 5x10^6 cells were seeded per 15 cm dish one day before starting the depletion experiment. UPF1 depletion was induced with 500 µM indole-3-acetic acid (IAA; Sigma-Aldrich; Cat# I5148) for 12 hours. All cells were harvested at the same time to minimize differences in cell numbers. Total RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research; Cat# R2052) including the recommended DNase I treatment according to the manufacturer's instructions.  The PacBio Kinnex full-length RNA sequencing library was constructed from four total RNA samples. First, each sample was processed into a PacBio barcoded Iso-Seq library, starting with 420 ng of total RNA and using the Iso-Seq Express 2.0 Kit (PacBio, #103-071-500). Then, 14 ng of each Iso-Seq library was additionally amplified to introduce Kinnex overhangs at the ends of the library molecules and pooled together to prepare one Kinnex library using the Kinnex Concatenation Kit (PacBio, #103-072-000).