cell line: CHO-k1 IgG-secreting cell line BC culture system: shake flask sampling time: day 4 culture system id: A3
Treatment protocol
Bioreactor batch culture conditions were kept at 37 oC with a working volume of 600 ml in a 1 litre DasGip parallel bioreactor systems (DasGip, an Eppendorf Company, Germany). pH was kept constant at 7.2 using CO2 sparging combined with 0.5 M NaOH. Agitation was kept at 100 rpm. Sparging with a mixture of oxygen and nitrogen was used to maintain the DO at 50 % air saturation. Total of 10 ppm concentration of Antifoam (Antifoam C Emulsion, Sigma-Aldrich®) was added to the bioreactors. Addition was done when needed. For the cultivation in a batch shake flask, the working volume was 60 ml in 250 ml total volume (VWR, USA) in an incubator (Multitron CO2 incubator; Infors HT) operated with 90% humidity, 8% CO2, and 37 oC at 100 rpm rotational speed and 50 mm orbital shaking diameter. Inocula for the batch shake flasks and the bioreactors were prepared from one pool of cells (shake flask). The starting cell density in both systems was 0.3 x106 cells ml−1. At the indicated time points, samples (3 ml) were taken from both the bioreactors and the shake flasks, centrifuged at 300 g for 10 minutes, after which the cell pellet was snap-frozen in liquid nitrogen and stored at -80 oC for later analysis.
Growth protocol
The CHO-k1 IgG-secreting cell line BC® was provided by Bioceros Holding BV, The Netherlands. All the cultures used chemically defined CD FortiCHO™ Medium (Gibco®, Life Technologies), supplemented with 4 mM L-glutamine and 0.5% (v/v) anti-clumping agent (both from Gibco®, Life Technologies). Pre-cultures were maintained in a 125 mL un-baffled shake flask (VWR, USA) with 20 ml working volume supplemented with selection reagents (200 µg/mL ZeocinTM and 5 µg/mL Blasticidin, both from Life technologies) for five passages prior to the experiments.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using RNeasy micro kit from Qiagen. RNA integrity was checked on chip analysis (Agilent 2100 bioanalyzer, Agilent Technologies, Amsterdam, the Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0).
Label
biotin
Label protocol
Purified total RNA (100ng per sample) was labeled with the Whole-Transcript Sense Target Assay (Affymetrix, Santa Clara, CA, USA; P/N 900652).
Hybridization protocol
Hybridization and washing of the Affymetrix CHO Gene 2.1 ST peg arrays were performed on a GeneTitan Instrument (Affymetrix, Santa Clara, CA) according to the manufacturer's recommendations.
Scan protocol
Arrays were scanned on an Affymetrix GeneTitan instrument (Affymetrix, Santa Clara, CA).
Data processing
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.40.2).
