For RNA-Seq and quantitative real-time PCR analyses, total RNA was extracted from three independent pneumococcus cultures grown in THY broth at 37°C grown at a mid-log-phase in the CO2 incubator. Briefly, the bacterial cultures were pelleted, washed, and adjusted to OD620 of 0.6. Next, 10 mL of these normalized cultures were pelleted and resuspended in 200 μL of the lysing buffer (PBS containing 25 μg/mL pneumo-phage lysin) (110 (link)) and further incubated for 1 h at 37 C for total lysis. The total RNA was extracted using the Norgen total RNA-extraction kit (Norgen Biotek Corp., Thorold, ON, Canada) per the manufacturer’s instructions. The DNA-free total RNA was obtained by treatment with the RNase-free DNase (Millipore). Qualitative and quantitative analyses of the total RNA were determined by the Agilent RNA6000 bioanalyzer (Agilent Technology, Santa Clara, CA). High-quality total RNA was determined based on RNA integrity (RIN) >7.
Norgen total rna extraction kit
Manufactured by Norgen Biotek
Sourced in Canada
The Norgen total RNA-extraction kit is a laboratory equipment designed for the isolation and purification of total RNA from various biological samples, including cells, tissues, and body fluids. The kit utilizes a silica-based membrane technology to capture and purify RNA molecules, ensuring high-quality RNA suitable for downstream applications such as RT-PCR, Northern blotting, and other RNA analysis techniques.
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Lab products found in correlation
2 protocols using norgen total rna extraction kit
1
RNA Extraction from Pneumococcus Cultures
2
RNA-Seq Analysis of Ascites Cells
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RNA was extracted from ascites cells using the Norgen Total RNA extraction kit (cat. # 17200, Norgen, Thorold, ON, Canada). Input was normalized based on cell count. RNA integrity numbers were measured using BioAnalyzer (Agilent, Santa Clara, CA, USA) and ranged from 7.1 to 9.9 (with one outlier of 2.9, which was removed from further analysis). RNA was quantified using Qubit (Thermofisher). Then, 800 ng of total RNA was used as input to the NEBNext Ultra RNA Library Prep Kit for Illumina using the polyA selection method (E7530S and E7490S). Libraries were barcoded using NEBNext multiplex oligos for Illumina (E7335S). Five samples per lane were pooled on an Illumina HiSeq 2500 flow cell and sequenced an average of 29M aligned reads per sample and an average quality score of 35, with 93% >Q30. Samples were processed using our published primary analysis tool, aRNApipe.16 (link) Count tables were processed using DESeq217 (link) for differential expression analysis. No clinical covariates were used in the model since the experiment was all done in vitro. R was used for all additional statistical analysis. Hierarchical clustering was done using the heatmap.2 function in the gplots R package.
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