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Sample GSM6723610

Query DataSets for GSM6723610

Status

Public on Jun 26, 2024

Title

ZN_D1_3

Sample type

SRA

Source name

CHOZN GS-/-

Organism

Cricetulus griseus

Characteristics

time: Day 1
cell line: CHOZN GS-/-
cell type: Chinese hamster ovary (CHO)
genotype: WT
phenotype: control, host cell line
replicate: rep 3

Treatment protocol

A cell culture volume not exceeding 5 million cells/mL was collected, spun down at 500 rpm for 5 min, aspirated, washed with 1X PBS, spun down at 500 rpm for 5 min, and aspirated. Cell pellets for RNA isolation were resuspended in 100 µL of RNALaterTM Stabilization Solution (Thermo Fisher Scientific, Cat. No. AM7021) and stored at -80˚C until ready for extraction.

Growth protocol

Cell cultures were maintained according to the CHOZN® Platform Technical Bulletin (Sigma-Aldrich) with some modifications. Both passage and fed-batch cultures were grown in Erlenmeyer shake flasks (VWR®, Cat. No. 89095-262) at 100 rpm, 37˚C, and 5% CO2. Each cell line was passaged at least three times post-thaw to ensure similarly aged, healthy cultures for subsequent transcriptomic analysis of fed-batch samples. For passages, the culture volume corresponding to 0.5 million cells/mL was spun for 5 min at 500 rpm, aspirated, washed with 1X phosphate buffered saline (PBS), spun for 5 min at 500 rpm, aspirated, and resuspended in 25 mL of fresh EX-CELL® CD CHO Fusion media (Sigma-Aldrich, Cat. No. 14365C). Fed-batch cultures were run in biological triplicate. Fed-batch cultures were passaged in the same way, but resuspended in 30 mL of fresh EX-CELL® Advanced™ CHO Fed-batch media (Sigma-Aldrich, Cat. No. 14366C). Both passage and fed-batch medium for the host cell line were supplemented with L-glutamine (Corning, Cat. No. MT25005CI) to a final concentration of 5 mM. Feedings always took place after sampling and were carried out according to the CHOZN® Platform Technical Bulletin. Briefly, the host cell line was fed L-glutamine to a final concentration of 5 mM starting on day 3 and on all remaining odd days. All cell lines were fed an EX-CELL® AdvancedTM CHO Feed 1 (Sigma-Aldrich, Cat. No. 24368C-1L) at 5% of the culture volume on day 3 and on all remaining odd days. Finally, all cell lines were fed D-(+)-Glucose solution (Sigma-Aldrich, Cat. No. G8769-100 mL) to a final concentration of approximately 4g/L daily, once the glucose concentration dropped below 2g/L.

Extracted molecule

total RNA

Extraction protocol

Extractions of RNA were carried out using the Qiagen RNeasy Plus Mini Kit (Qiagen, Cat. No. 74136). Extractions were performed according to the manufacturer’s protocol for “Purification of Total RNA from Animal Cells” with the following changes: samples were prepared per treatment protocol above, option 3c was followed for Step 3, molecular grade ethanol was used for Step 5, optional Step 10 was used, and Dnase/Rnase-free water was heated to 37 ˚C and allowed to incubate on the column for 5 min for elution. Quantification of RNA was performed by measuring absorbance at 260 and 280 nm using a nanospectrophotometer. Multiple aliquots of 100 ng/µL were prepared and stored at -80 ˚C until ready for analysis. Concentrations of RNA and RNA Integrity Numbers (RIN) were determined by Qubit fluorometer (Invitrogen) and 2100 Bioanalyzer (Agilent), respectively.
After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. For prokaryotic organisms or eukaryotic organisms' long-non-coding libraries, rRNA is removed using the Ribo-Zero kit that leaves the mRNA. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment.
The quality control of library consists of three steps:
(1) Qubit 2.0: tests the library concentration preliminarily.
(2) Agilent 2100: tests the insert size.
(3) Q-PCR: quantifies the library effective concentration precisely.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

no count file generated

Data processing

Data processing was performed using the Palmetto Cluster, R, and Python
Trimmomatic (0.36) for adapter removal
FastQC (0.11.8) for quality control analysis of raw reads
STAR (2.6.0a) for alignment of the raw reads to the CHO CriGri-PICR reference genome assembly (GCF_003668045.1_CriGri-PICR)
HTSeq (0.10.0) for generation of the read count tables
To generate read counts for the three product mRNAs, the CriGri-PICR genome file was amended to include three additional gene sequence files for the products: IgG1 light chain (IgG1 LC), IgG1 heavy chain (IgG1 HC), and EPO-Fc.
Temporal expression profile groups were generated through hierarchical clustering using the DEGreport (1.30.0) package after performing the likelihood ratio test (LRT) on a reduced DESeq2 (1.34.0) model. The full model included variables denoting cell line, time, and the interaction between cell line and time. The interaction variable was removed in the reduced model to test for variance due to cell line over time. We analyzed 2500 genes with the lowest adjusted p-values, with 0.01 as the upper cutoff limit.
The clusterProfiler package (4.2.2) was used to conduct Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway and Gene Ontology (GO) enrichment on the obtained temporal expression cluster profiles. The organism identifier “cge” was used to denote Chinese hamster KEGG pathway enrichment. The Chinese hamster annotation AH96228, available through Annotation Hub, was used for GO enrichment categories. Enrichment analyses were calculated using an adjusted p-value cutoff of 0.05.
Principal component analysis (PCA) and differential expression testing based on the Wald statistical pairwise test was conducted using DESeq2 (1.34.0). Fragments per kilobase pair million (FPKM) were calculated using the built-in DESeq2 function based on the known lengths of target genes. For all pairwise differential gene expression analysis (DGEA), the absolute value of the log2 fold change (log2FC) cutoff was set to 0.5 and an adjusted p-value cutoff of 0.01 was used, as identified by an elbow of all DEGs versus various adjusted p-value cutoffs.
Assembly: CHO CriGri-PICR reference genome assembly (GCF_003668045.1_CriGri-PICR)
Supplementary files format and content: Text file of read counts generated for each geneID (RefSeq format) for a specific sample (e.g. ZN_D0_1)

Submission date

Nov 09, 2022

Last update date

Jun 26, 2024

Contact name

Mark A. Blenner

E-mail(s)

[email protected]

Organization name

University of Delaware

Department

Chemical and Biomolecular Engineering