HT-1376 cells were seeded in T25 flasks overnight. The next day media was replaced with normal media without IFN (mock). Total RNA was prepared from cells 24 hrs post-treatment using the RNeasy Mini Kit (Qiagen). PolyA+ RNA was enriched from total RNA using the Dynabeads mRNA Purification Kit (Invitrogen). cDNA libraries were prepared from 200 ng of PolyA+ RNA using the Direct cDNA Sequencing Kit (Oxford Nanopore), according to the PCR-free 1D read protocol for full-length cDNA (Oxford Nanopore, SQK-DCS109), with some modifications. Specifically, RNase Cocktail Enzyme Mix (ThermoFisher) was used during the RNA digestion step after the first-strand synthesis; all reaction amounts for reverse transcription reactions up to the second-strand synthesis step were doubled; from second-strand synthesis up to adapter ligation, reactions were 1.5x of original amounts; during adapter ligation, 35 ul Blunt/TA Ligase Master Mix was used instead of the recommended 50 ul, and nuclease-free water was excluded. Final libraries were loaded into MinION Fluidics Module flow cells (Oxford Nanopore, FLO-MIN106D), and sequencing was carried out on GridION MK1 and MinION MK1C instruments (Oxford Nanopore) for 3 days, using default parameters.
isolate
caucasian
age
58
biomaterial_provider
ATCC
sex
female
tissue
Urinary bladder
cell_line
HT-1376
cell_subtype
Bladder carcinoma
cell_type
Epithelial cells
disease
Carcinoma
ethnicity
Caucasian
race
White
treatment
Mock
BioSampleModel
Human