title: 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
design: 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
library_name: HCT_N_AID_UPF1_12h_IAA_ONT_dRNA_2_s
library_construction_protocol: 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. Poly(A) mRNA was purified from total RNA using the Dynabeads™ mRNA Purification Kit (Invitrogen; Cat#61006). After one round of purification according to the manufacturer's instruction, the samples were incubated for a second round of binding to the same beads, repeating the protocol for a second time after the rebinding of the RNA to the beads. Successful enrichment for poly(A) RNA was confirmed by High Sensitivity RNA ScreenTape Analysis. ONT direct RNA sequencing libraries were prepared from 300 ng of poly(A) RNA using the Direct RNA Sequencing Kit (Oxford Nanopore Technologies, #SQK-RNA004) following the manufacturer's instructions.
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-14725_2:HCT_N_AID_UPF1_12h_IAA_ONT_dRNA_2_s