NextSeq 500 sequencing; Adherent and suspension baby hamster kidney cells have a different cytoskeleton and surface receptor repertoire
Adherent and suspension baby hamster kidney cells have a different cytoskeleton and surface receptor repertoire
Batches of each of the cell lines BHK179, BHK-2P and BHK-InV were lysed in three volumes of TRIzol LS Reagent. Culture conditions were 37 °C, 5% CO2, and for suspension cells 320 rpm in TubeSpin bioreactors (TPP Techno Plastic Products AG, Trasadingen, Switzerland) at 80% relative humidity. All adherent cell lines were cultured in Minimum Essential Medium Eagle (MEM) supplemented with Hanks' and Earle's salts (Sigma-Aldrich) with 10% FBS. All suspension cell lines were adapted to grow in CellventoTM BHK200 medium (Merck KGaA). After adding trichloromethane the aqueous phase was collected and mixed with one volume of 100% ethanol. From this, total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) with on-column DNase digestion with the RNase-Free DNase Set (Qiagen). The ERCC RNA Spike-In Mix 1 (Thermo Fisher Scientific) was added and used as an internal control for all following steps. Next, polyadenylated mRNA was isolated from 1-3 µg of high-quality total RNA using the Dynabeads mRNA DIRECT Micro kit (Thermo Fisher Scientific).  The mRNA was subsequently fragmented and used for cDNA synthesis and library preparation using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, USA) according to the manufacturer's instructions.