RNA-seq of Gallus gallus
Four independent batches (n = 4) of anchorage-dependent cells were adapted to suspension for RNA sequencing analysis. Cell lysate from each time point was stored at 80 C until all samples had been collected. RNA was extracted from simultaneously from all samples to minimize batch effects. RNA was isolated from cell lysate and purified from DF-1s at the previously specified time points using RNeasy Mini Kit (Qiagen, Hilden, Germany; 74104) according to manufacturers instructions. Adherent cells were dissociated from tissue culture plastic using TrypLE to avoid RNA degradation before purification[88]. RNA concentration, 260/280 ratios, and 260/230 ratios were determined using NanoDrop 2000 Spectrophotometer (ThermoFisher, ND-2000).RNA samples were sent on dry ice to Novogene (Sacramento, California, USA) for library preparation and RNA sequencing. Sample quality of RNA was determined by Novogene using gel electrophoresis, Nanodrop, and Agilent2100 to determine RNA Integrating Number (RIN). Non-directional library preparation was performed and libraries were sequenced on an Illumina NovaSeq X Plus using a 25B flowcell with paired-end, 150 base pair (bp) reads.
SUB15651510
T5R2