|
| Status |
Public on Feb 12, 2018 |
| Title |
HP biological replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
Chinese hamster ovary cells
|
| Organism |
Cricetulus griseus |
| Characteristics |
parental strain: CHOZN® GS-/-
parental strain: CHO cells
productivity: high producer
ercc: Mix1
|
| Growth protocol |
Separate vials were thawed for each biological replicate and passaged for two weeks prior to bioreactor inoculation. Cells were seeded at 0.5 million cells/mL in a 1L Mini-Bioreactor (Applikon Biotechnologies) with a working volume of 700 mL of EX-CELL® CD CHO Fusion Medium (Sigma Aldrich) containing 25 µM L-Methionine sulfoximine (MSX) as selection. The bioreactor was operated at 125 rpm stirring speed, 37°C, pH 6.9 and dissolved oxygen at 50% air saturation. Glucose concentration was determined using a BioProfile FLEX (Nova Biomedical) and a bolus of 3 g/L of glucose was added to the culture when the extracellular glucose reached 2 g/L, thus maintaining the glucose level between 2 and 5 g/L.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For the high producer (HP) and low producer (LP) mRNA-Seq samples were prepared with Illumina's TruSeq RNA Sample Prep Kit v2, as per manufacturer’s protocol, using 1 µg of total RNA as input. The samples were amplified using 12 cycles of PCR and the libraries were then loaded onto the channels of the flow cell at 11 pM concentration, multiplexing 3 samples per lane. Sequencing was performed on the Hiseq2000 using TruSeq v3 SBS reagents and 100 bp paired-end reads.
Five million cells were harvested in the mid-exponential phase from each bioreactor, i.e. three biological replicates per cell line. Cells were centrifuged at 500g for 3 min, and the cell pellet was frozen in dry ice and stored at -80 °C. RNA was extracted from the ice thawed pellet using the RNeasy Mini Kit (Qiagen) as per manufacturer’s instructions for animal cells using spin technology. RNA concentration was measured with a Qubit fluorometer (Life Technologies) and the quality was evaluated with an Agilent 2100 bioanalyzer using a RNA Nano Chip (Agilent Technologies). ERCC ExFold RNA Spike-In Mixes (Life Technologies) were added to the RNA samples to assess the accuracy of measurements of differential gene expression on the platform used. Mix 1 or Mix 2 were added to the different samples to compare the expected versus observed fold-change ratio (Mix 1:Mix 2).
RNA libraries were prepared for sequencing using standard Illumina protocols (described for each sample)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Gene expression HP vs LP.xlsx
|
| Data processing |
The reads were trimmed to 75 bases before mapping in order to improve their quality.
TopHat open-source software (version 2.0.12, using Bowtie version 2.2.3.0 and SAMtools version 0.1.19.0), was used with default settings to align the reads to the CHO-K1 genome. A combined file containing the CHO-K1 genomic scaffolds (CriGri_1.0), the mitochondrial sequence (NC_007936.1) and the ERCC standards sequences was used as a reference.
Cufflinks open-source software (version 2.2.1) was used to assemble transcripts, estimate their abundance and tests for differential expression. The annotated version of the CHO-K1 genome last modified on 2014/08/11 was used. Cuffdiff was run using default setting and the frag-bias-correct (for bias detection and correction) and the multi-read-correct (to more accurately weight reads mapping to multiple locations in the genome) options to improve the accuracy of abundance estimates. The genes were classified as differentially expressed if the adjusted p-value (q-value) was lower than 0.05.
Genome_build: A combined file containing the CHO-K1 genomic scaffolds (CriGri_1.0), the mitochondrial sequence (NC_007936.1) and the ERCC standards sequences was used as a reference.
Supplementary_files_format_and_content: Two excel files containing Cuffdiff differential expression analysis
|
| |
|
| Submission date |
Jul 07, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Camila Antonia Orellana |
| E-mail(s) |
c.orellana@uq.edu.au
|
| Organization name |
The University of Queensland
|
| Department |
Australian Institute for Bioengineering and Nanotechnology
|
| Street address |
Crn College and Cooper Rds (Bldg 75)
|
| City |
Brisbane |
| State/province |
Queensland |
| ZIP/Postal code |
4072 |
| Country |
Australia |
| |
|
| Platform ID |
GPL19001 |
| Series (1) |
| GSE100908 |
High technical resolution transcriptomics analysis for CHO cells |
|
| Relations |
| BioSample |
SAMN07334560 |
| SRA |
SRX2991084 |
