For transcriptomics 0.5 mL culture was transferred into reaction tubes and centrifuged at 11.000 × g for 2 min, and the pellet was frozen in liquid nitrogen. The total RNA of the cells was isolated using the Total RNA Isolation Mini Kit (Agilent, Santa Clara, CA). The integrity of the RNA was measured using the BioAnalyzer Pico-Kit (Agilent, Santa Clara, CA). RNA-sequencing was performed by the Max Planck-Genome-Centre Cologne, Germany (https://mpgc.mpipz.mpg.de/home/ ). The sequencing reads were analyzed and mapped using the CLC Software (QIAGEN, Venlo, NL). For normalization, gene expression was calculated as transcripts per kilobase million (TPMs). RNA was sampled four times at the first time point t0; and two samples were collected at the remaining time points. For the time points t13, t15, t19 and t24 one of the two replicates was excluded due to low quality of the sampled RNA. Transcriptomics metadata is accessible under the GEO number GSE131992.
Bioanalyzer pico kit
Manufactured by Agilent Technologies
The Bioanalyzer Pico Kit is a laboratory instrument designed for the analysis of DNA, RNA, and protein samples. It provides automated electrophoresis and detection of these biomolecules in a microfluidic chip format.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000 system, by Illumina (2 mentions)
Trio rna seq library preparation kit, by Tecan (2 mentions)
Total rna isolation mini kit, by Agilent Technologies (1 mentions)
Novaseq s4 lane, by Illumina (1 mentions)
Quantseq 3 mrna seq kit, by Lexogen (1 mentions)
Ingenuity pathway analysis (ipa), by Qiagen (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
4 protocols using bioanalyzer pico kit
1
Transcriptomic Profiling Using RNA-Seq
2
Density Fractionation and Differential Sedimentation RNA-seq
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RNA-seq libraries for the density fractionation were generated using 1 μg of RNA as starting material and depleting ribosomal RNA using Ribocop V3 (Lexogen). Post ribosomal RNA depletion, RNA content was measured with a Bioanalyzer pico kit (Agilent). Then 1% RNA spike-in RNA variants (SIRVs) were spiked-in (Lexogen), and RNA-seq libraries were generated and amplified using the CORALL RNA-seq kit (Lexogen) according to the manufacturer’s instructions. All CORALL-generated libraries were sequenced in parallel on four Novaseq S4 lanes (Illumina). RNA-seq libraries for the differential sedimentation speed based cell fractionation experiment were performed using QuantSeq 3′ mRNA-Seq kit (Lexogen) according to manufacturer’s instructions. A total of 400 ng of total RNA and 0.1% RNA SIRVs (Lexogen) were used for library preparation of each fraction. All libraries were balanced, multiplexed and pooled, and run on two single-end Novaseq SP lanes (Illumina).
3
Transcriptomic Analysis of TRAP Samples
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RNA recovered from TRAP was sequenced and has been uploaded to the Sequence Read Archive (National Center for Biotechnology Information; Accession code, PRJNA597018; Forbes et al., 2020 (link)). Briefly, sequencing libraries were prepared using the Trio RNA-Seq Library Preparation Kit (NuGen) for all samples with a RNA Integrity Number value > 7 (Bioanalyzer Pico Kit, Agilent). Paired-end reads (50 bp) were obtained using the NovaSeq 6000 System (Illumina), and trimmed with Trimmomatic (Bolger et al., 2014 (link)) according to the Library Preparation Kit protocol (NuGen). Reads were mapped to the mouse reference genome (Genome Reference Consortium Mouse Build 38, mm10) using STAR (Dobin et al., 2013 (link)), and read counts were generated using Htseq (Anders et al., 2015 (link)). Normalized read counts were generated using DESeq2 package for R (Love et al., 2014 (link)).
4
RNA-Seq Analysis of Hypoxia Response
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RNA samples were sent to the Genome Sciences and Bioinformatics core at Penn State College of Medicine for Library preparation and RNA-sequencing. RNA quality was determined using the Bioanalyzer Pico Kit (Agilent), and quantity was determined with the Qubit fluorometer (ThermoFisher). Libraries were prepared from RNA (RIN > 7) using the Trio RNA-Seq Library Preparation Kit (NuGen), according to the manufacturer’s protocol. Libraries were sequenced using the NovaSeq 6000 System (Illumina) to obtain 50 base pair (bp), paired-end reads. The first 5 bp were trimmed from the 5′ end of the reads using Trimmomatic78 (link), as recommended by the Library Preparation Kit. Reads were mapped to the mouse reference genome (Genome Reference Consortium Mouse Build 38 – mm10) using STAR79 (link), and read counts were generated using HTseq80 (link). Differential gene expression and normalized read counts were generated using DESeq2 package for R81 (link). DEGs reported have normalized read counts above 5 in all samples, and an adjusted p < 0.05 (Wald test with Benjamini−Hochberg post hoc). Ingenuity Pathway Analysis (Qiagen) was used to identify cellular and molecular functions and the associated genes implicated from DEGs between the HX and HX-EE groups.
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