All CHO cell lines were cultivated at 37 °C, 7% CO2 and humidified atmosphere. K1-STAT-4-FCS-HOST (CHO-K1, ECACC-CCL61) and DUKXB11-STAT-4-FCS-HOST (CHO-DUKXB11, ATCC CRL-9096) were grown in DMEM/Ham’s F12 media (1:1, Merck KGaA, Darmstadt, Germany) supplied with 5% fetal calf serum (GE Healthcare, Little Chalfont, UK) and 4 mM L-Gln. DUKXB11-STAT-4-FCS-HOST cells were additionally supplied with 1x hypoxanthine/thymidine (HT) supplement (Thermo Fisher Scientific, Waltham, MA). DUKXB11-SHAKE-4-SF-HOST (suspension and serum-free adapted CHO-DUKXB11) cells were cultivated in DMEM/Ham’s F12 (1:1), supplemented with 4 mM L-Gln, 0.25% soy peptone, 0.1% Pluronic F68 (BASF, Germany), 1x protein free supplement (Polymun Scientific, Austria) and 1x HT supplement. K1-SHAKE-8-SF-HOST/K1-FERM-8-SF-HOST (suspension and serum-free adapted CHO-K1), K1-SHAKE-0-SF-HOST/K1-FERM-0-SF-HOST (suspension, serum-free and L-Gln-free adapted CHO-K1), S-SHAKE-8-SF-HOST (CHO-S), DUKXB11-SHAKE-0-SF-PROD/DUKXB11-FERM-0-SF-PROD-M17/DUKXB11-0-SF-FERM-PROD-MNC (recombinant suspension and serum-free adapted CHO-DUKXB11, producing an Erythropoietin-Fc fusion protein) were grown in CD CHO (Thermo Fisher Scientific), supplemented with 1x Anti-Clumping Agent (Thermo Fisher Scientific). K1-SHAKE-8-SF-HOST/K1-FERM-8-SF-HOST and S-SHAKE-8-SF-HOST were additionally supplied with 8 mM L-Gln. DUKXB11-SHAKE-0-SF-PROD, DUKXB11-FERM-0-SF-PROD-M17 and DUKXB11-0-SF-FERM-PROD-MNC cells were supplied with 0.19 µM methotrexate. DUKXB11-FERM-0-SF-PROD-M17 additionally harbours a vector overexpressing miR-17, while DUKXB11-FERM-0-SF-PROD-MNC harbours an empty vector control. DUKXB11-SHAKE-4-SF-HOST, K1-SHAKE-8-SF-HOST, K1-SHAKE-0-SF-HOST, S-SHAKE-8-SF-HOST and DUKXB11-SHAKE-0-SF-PROD were cultivated in shaker flasks at 140 revolutions per minute and a 25 mm shaking diameter. K1-FERM-8-SF-HOST, K1-FERM-0-SF-HOST, DUKXB11-FERM-0-SF-PROD-M17 and DUKXB11-FERM-0-SF-PROD-MNC were cultivated in DASGIP bioreactor system (DASGIP AG, Jülich, Germany) controlled by DASGIP Control 4.0 software with similar settings as described before (Taschwer et al., 2012). Briefly, advanced spinners vessels used for the cultivation with 800 ml working volume and magnetic drive and pitch blade impellers for constant stirring (80 rpm). Temperature was maintained at 37 °C, constant 30% oxygen concentration was held via probe (Broadley James Oxyprobe) by air saturation and neutral pH (Mettler Toledo) was regulated with 0.5 M NaOH and CO2.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using the phenol-chloroform extraction method. Adherent CHO cell lines were washed with PBS and detached using Trypsin. Adherent and suspension cells were collected by centrifugation and lysed using TRI reagent (Sigma- Aldrich, St. Louis, MO). RNA isolation was performed according to the manufacturer’s recommendations. In brief, phase separation was conducted by the addition of chloroform, and the aqueous phase was collected. After centrifugation and washing with 2-Propanol and 70 % Ethanol, RNA pellets were air-dried, and resuspended in nuclease-free water (NFW). For quantity and purity estimation, absorbances at 230, 260 and 280 nm were measured using a NanoDrop ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific). Total RNA quality was assessed on the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) using the RNA 6000 Nano Kit (Agilent Technologies). Only RNA with a RNA integrity number (RIN) > 7 were used for microarray hybridization.
Label
Cy3
Label protocol
End-labeling of 800 ng of total RNA was performed using the Exiqon Power Labeling Kit (Exiqon, Denmark) together with synthetic spike-in controls according to the instructions by the manufacturer.
cho cell line: DUKXB-11, CHO-K1, CHO-S growth phase: not applicable growth rate: not applicable
Treatment protocol
No treatments were performed
Growth protocol
All CHO cell lines were cultivated at 37 °C, 7% CO2 and humidified atmosphere. K1-STAT-4-FCS-HOST (CHO-K1, ECACC-CCL61) and DUKXB11-STAT-4-FCS-HOST (CHO-DUKXB11, ATCC CRL-9096) were grown in DMEM/Ham’s F12 media (1:1, Merck KGaA, Darmstadt, Germany) supplied with 5% fetal calf serum (GE Healthcare, Little Chalfont, UK) and 4 mM L-Gln. DUKXB11-STAT-4-FCS-HOST cells were additionally supplied with 1x hypoxanthine/thymidine (HT) supplement (Thermo Fisher Scientific, Waltham, MA). DUKXB11-SHAKE-4-SF-HOST (suspension and serum-free adapted CHO-DUKXB11) cells were cultivated in DMEM/Ham’s F12 (1:1), supplemented with 4 mM L-Gln, 0.25% soy peptone, 0.1% Pluronic F68 (BASF, Germany), 1x protein free supplement (Polymun Scientific, Austria) and 1x HT supplement. K1-SHAKE-8-SF-HOST/K1-FERM-8-SF-HOST (suspension and serum-free adapted CHO-K1), K1-SHAKE-0-SF-HOST/K1-FERM-0-SF-HOST (suspension, serum-free and L-Gln-free adapted CHO-K1), S-SHAKE-8-SF-HOST (CHO-S), DUKXB11-SHAKE-0-SF-PROD/DUKXB11-FERM-0-SF-PROD-M17/DUKXB11-0-SF-FERM-PROD-MNC (recombinant suspension and serum-free adapted CHO-DUKXB11, producing an Erythropoietin-Fc fusion protein) were grown in CD CHO (Thermo Fisher Scientific), supplemented with 1x Anti-Clumping Agent (Thermo Fisher Scientific). K1-SHAKE-8-SF-HOST/K1-FERM-8-SF-HOST and S-SHAKE-8-SF-HOST were additionally supplied with 8 mM L-Gln. DUKXB11-SHAKE-0-SF-PROD, DUKXB11-FERM-0-SF-PROD-M17 and DUKXB11-0-SF-FERM-PROD-MNC cells were supplied with 0.19 µM methotrexate. DUKXB11-FERM-0-SF-PROD-M17 additionally harbours a vector overexpressing miR-17, while DUKXB11-FERM-0-SF-PROD-MNC harbours an empty vector control. DUKXB11-SHAKE-4-SF-HOST, K1-SHAKE-8-SF-HOST, K1-SHAKE-0-SF-HOST, S-SHAKE-8-SF-HOST and DUKXB11-SHAKE-0-SF-PROD were cultivated in shaker flasks at 140 revolutions per minute and a 25 mm shaking diameter. K1-FERM-8-SF-HOST, K1-FERM-0-SF-HOST, DUKXB11-FERM-0-SF-PROD-M17 and DUKXB11-FERM-0-SF-PROD-MNC were cultivated in DASGIP bioreactor system (DASGIP AG, Jülich, Germany) controlled by DASGIP Control 4.0 software with similar settings as described before (Taschwer et al., 2012). Briefly, advanced spinners vessels used for the cultivation with 800 ml working volume and magnetic drive and pitch blade impellers for constant stirring (80 rpm). Temperature was maintained at 37 °C, constant 30% oxygen concentration was held via probe (Broadley James Oxyprobe) by air saturation and neutral pH (Mettler Toledo) was regulated with 0.5 M NaOH and CO2.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using the phenol-chloroform extraction method. Adherent CHO cell lines were washed with PBS and detached using Trypsin. Adherent and suspension cells were collected by centrifugation and lysed using TRI reagent (Sigma- Aldrich, St. Louis, MO). RNA isolation was performed according to the manufacturer’s recommendations. In brief, phase separation was conducted by the addition of chloroform, and the aqueous phase was collected. After centrifugation and washing with 2-Propanol and 70 % Ethanol, RNA pellets were air-dried, and resuspended in nuclease-free water (NFW). For quantity and purity estimation, absorbances at 230, 260 and 280 nm were measured using a NanoDrop ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific). Total RNA quality was assessed on the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) using the RNA 6000 Nano Kit (Agilent Technologies). Only RNA with a RNA integrity number (RIN) > 7 were used for microarray hybridization.
Label
Cy5
Label protocol
End-labeling of 800 ng of total RNA was performed using the Exiqon Power Labeling Kit (Exiqon, Denmark) together with synthetic spike-in controls according to the instructions by the manufacturer.
Hybridization protocol
Slides were hybridized over night at 56°C in a Tecan HS 400 hybridization station, followed by automated washing and drying with nitrogen (Tecan, Austria).
Scan protocol
Immediately after drying, arrays were scanned using the Roche Nimblegen MS200 scanner (Roche, Germany) at 10 µM resolution and auto-gain settings.
Data processing
Feature extraction from the generated images was performed using GenePix 4.1 (Molecular Devices, Sunnyvale,CA). The resulting GPR-files were processed in R/Bioconductor using the LIMMA package (Ritchie et al., 2015). Raw data of previously published arrays (Gene Expression Omnibus accession number: GSE52994) was compiled with the here generated data for jointly analysis. Global LOESS normalization and Normexp background correction were performed, and the log2-fold changes of the miRNAs were calculated against the common reference pool (LogFC) for each array. To be considered expressed, the average probe intensity had to exceed the average background intensity plus two times the standard deviation. The probe’s validity was reviewed by updating the corresponding miRNA accessions to miRBase v21.