direct cDNA-seq of human KTC-1 cell line
Human cell line KTC-1 (human papillary thyroid cancer cell line) was grown to 85-90% confluence in 10cm dishes in RPMI media supplemented with 10% fetal bovine serum utilizing standard techniques. The cells were rinsed twice with cold, sterile PBS after which 700 l TRIzol reagent (Life Technologies) was added. Following incubation for 5 min at room temperature, the cells were collected and mixed with 700 l 100% ethanol. RNA isolation was performed with the Direct-Zol RNA mini prep Kit (Zymo Research) as per manufacturers instructions. Poly(A)-tailing reaction using E. coli EPAP (NEB# M0276) was designed to perform in a 26 l reaction (reaction size can be scaled up as needed). A total of 3000 ng total RNA was used to prepare dcDNA-seq library. The dcDNA library was produced using the SQK-DCS108 kit (ONT, Oxford, UK) which includes an RT step. Direct sequencing was performed on a single R9.5/FLO-MIN107 flow cell.