Monoclonal RPE1(Cas9) cells were harvested at 90% confluency and total RNA was purified using the QIAGEN RNeasy Plus Mini Kit (Cat No.: 74134), according to the manufacturer's protocol. RNA was stored at -80°C and analyzed by RNA-Seq in quadruplicates. For library preparation, total RNA was quantified using the Qubit 2.0 fluorometric assay (Thermo Fisher Scientific). Sequencing libraries were prepared from 125 ng of total RNA using a 3'DGE mRNA-seq research grade sequencing service (Next Generation Diagnostics srl) which included library preparation, quality assessment and sequencing on a NovaSeq 6000 sequencing system using a single-end, 100 cycle strategy (Illumina Inc.) (46 (link)). The bioinformatics workflow included analysis of raw data by Next Generation Diagnostics srl proprietary 3'DGE mRNA-seq pipeline (v2.0) which involves a cleaning step by quality filtering and trimming, alignment to the reference genome and counting by gene (47–49 (link)). We filtered out all genes having < 1 cpm in less than n_min samples and Perc MM reads > 20% simultaneously. Differential expression analysis was performed using edgeR (Supplementary Table S7 ) (50 (link)).
Qubit 2.0 fluorometric assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Qubit 2.0 fluorometric assay is a laboratory instrument designed for the quantification of DNA, RNA, and protein samples. It utilizes fluorescent dyes that bind specifically to the target molecules, allowing for accurate and sensitive measurement of their concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000 sequencing system, by Illumina (2 mentions)
Dneasy powersoil dna isolation kit, by Qiagen (2 mentions)
Nuclease free water, by Thermo Fisher Scientific (2 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Quant it picogreen kit, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyser, by Agilent Technologies (1 mentions)
Rneasy micro mini plus kit, by Qiagen (1 mentions)
Rlt plus lysis buffer, by Qiagen (1 mentions)
5 protocols using qubit 2.0 fluorometric assay
1
Transcriptome Analysis of RPE1 Cells
2
Comprehensive oocyte cDNA analysis
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The amplified cDNA of oocytes and cumulus cells was subjected to quality and quantity analysis using the Qubit® 2.0 fluorometric assay (Thermofisher, UK), the 2100 Bioanalyser (Agilent Technology, UK), the PicoGreen measurement (Quant-it PicoGreen Kit, ThermoFisher, cat. no. P7589) and the TapeStation assay (Agilent, UK). A minimum cDNA concentration of 0.2 ng/µl was required together with a high-quality amplification profile.
3
RNA-seq Analysis of Pluripotent Stem Cells and Astrocytes
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Four to five biological replicates per cell type of human pluripotent stem cells, human primary cortical astrocytes, and human induced astrocytes (>day 30 of differentiation) were harvested in RLTplus Lysis buffer (Qiagen, 1053393). Total RNA was isolated using the RNeasy micro/mini plus kit (Qiagen, 74034) and stored at -80°C. Total RNA was quantified using the Qubit 2.0 fluorometric Assay (Thermo Fisher Scientific). Libraries were prepared from 125 ng of total RNA using a 3′DGE mRNA-seq research grade sequencing service (Next Generation Diagnostic srl)50 (link) which included library preparation, quality assessment and sequencing on a NovaSeq 6000 sequencing system using a single-end, 100 cycle strategy (Illumina Inc.). The raw data were analyzed by Next Generation Diagnostics srl proprietary 3′DGE mRNA-seq pipeline (v1.0) which involves a cleaning step by quality filtering and trimming, alignment to the reference genome and counting by gene.51 (link),52 (link) Differential expression analysis was performed using edgeR.53 (link) Samples were sequenced and analyzed at TIGEM (Pozzuoli, Italy).
4
Faecal DNA Extraction for Microbiome
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Microbial DNA was extracted from faecal pellets using the DNeasy PowerSoil® DNA isolation kit (QIAGEN) as per the manufacturer’s instructions. Briefly, 0.25 grams (g) of faeces (frozen) was added to the 750 microliters (µl) bead bed, 100 µl of sterilised milli-Q ultra-pure water and 160 µl of the C1 solution were added and beat-beating was performed by vortexing the tubes on high for 10 minutes (min). Final elution was performed with 100 µl of sterile nuclease-free water (Invitrogen, Mulgrave, VIC, Australia). DNA concentration was quantified by Qubit 2.0 Fluorometric assay (Life Technologies, Carlsbad, CA, USA), and adjusted to a concentration of 10 ng/μl.
5
Faecal DNA Extraction Protocol
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Microbial DNA was extracted from faecal pellets using the DNeasy PowerSoil® DNA isolation kit (QIAGEN) as per the manufacturer's instructions. Brie y, 0.25 grams (g) of faeces (frozen) was added to the 750 microliters (µl) bead bed, 100 µl of sterilised milli-Q ultra-pure water and 160 µl of the C1 solution were added, the tubes were vortexed on high for 10 minutes (min). Final elution was performed with 100 µl of sterile nuclease-free water (Invitrogen, Mulgrave, VIC, Australia). DNA concentration was quanti ed by Qubit 2.0 Fluorometric assay (Life Technologies, Carlsbad, California, USA), and adjusted to a concentration of 10 ng/µl.
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