Total RNA from purified N, ACT and nTH21 spleen cell pools were extracted with Qiagen RNeasy Mini Kits (Qiagen, Hilden, Germany). Poly-A-enriched mRNA was reverse transcribed and amplified using the NuGEN Ovation Kit (NuGEN, San Carlos, CA, USA). Paired-end cDNA was sequenced with an Illumina MiSeq at 106 base pair length (Illumina, San Diego, CA, USA). Reads were checked with FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit ), trimmed with Trimmomatic, and aligned to the GRCm38.73 assembly transcriptome with Bowtie. Transcript expression levels were estimated in transcripts per million (TPM) using RSEM (Li and Dewey, 2011 (link)). Differential expression analysis across the sorted populations was conducted with EBSeq (Leng et al., 2013 (link)). Detailed analytical procedures are described in Supplemental Experimental Procedures
Nugen ovation kit
Manufactured by Tecan
Sourced in United States
The Nugen Ovation kit is a lab equipment product designed for RNA amplification. It provides a method for generating high-quality amplified RNA from small amounts of input RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Gene ontology amigo term enrichment tool, by Qiagen (2 mentions)
Rnaexpress application v 1, by Illumina (2 mentions)
Ingenuity pathway analysis (ipa), by Qiagen (2 mentions)
Rneasy plus kit, by Qiagen (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Pen slides, by Zeiss (1 mentions)
5 protocols using nugen ovation kit
1
RNA-seq Analysis of Immune Cell Subsets
2
RNA-seq Analysis of Islet Samples
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RNA samples from three mice per group were used for RNA-seq (ESM Table 1 ). Total RNA was extracted from islet samples (176 ± 11 islets per mouse) using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA). RNA integrity was confirmed using the Agilent Bioanalyzer 2100 (RNA integrity number [RIN] range: 6.8–8.9). RNA-seq libraries were prepared using the TruSeq with Ribo-Zero treatment (Illumina, San Diego, CA, USA) to deplete ribosomal RNA. cDNA libraries were generated using the NuGEN Ovation kit (NuGEN, Redwood City, CA, USA). Illumina’s NextSeq 500 platform was used to generate 75 bp, paired-end reads (64 ± 1.4 million reads per sample). Short reads were aligned to the mouse reference genome build GRCm38 (mm10) using the Spliced Transcripts Alignment to a Reference software (STAR aligner) [18 (link)]. Between 65% and 75% (mean 70%) of the reads mapped uniquely to the mouse genome. The HT-Seq package [19 (link)] was used to count the number of fragments aligned to known exonic regions. Gene expression was measured as total fragment counts per gene. Sample clustering of islet RNA-seq using multidimensional scaling largely reflected genotype (ESM Fig. 2 ).
3
RNA-Seq Analysis of CD4+ T Cells
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RNA was prepared from CD4+ T cells using the QIAgen RNeasy Plus Kit. The Gladstone Institutes Genomics Core carried out the downstream processing of the RNA samples. Strand-specific cDNA libraries were prepared using the Nugen Ovation kit (Nugen) and the libraries were deep sequenced on NextSeq 500 using single-end 75 bp sequencing. RNA-seq analysis was done using the Illumina RNAexpress application v 1.1.0. Briefly, alignment of RNA-Seq reads was performed with the STAR aligner and, after alignment of aligned reads to genes, differential gene expression was analyzed with DESeq2. Pathway analysis was carried out using Ingenuity Pathway Analysis (QIAGEN) and Gene Ontology AmiGO Term Enrichment tool (Biological Processes and Cellular Components). For the Ingenuity analysis, the entire dataset was uploaded, and a two-direction analysis carried using filters to restrict the analysis to differentially expressed genes as above. Refer to the manufacturer’s website (http://www.ingenuity.com ) for further details. For Gene Ontology, individual lists of genes were uploaded and analyzed separately. Venn diagrams were generated using the online tool: http://genevenn.sourceforge.net/ .
4
Periocular Mesenchymal Tissue RNA-Seq
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Freshly harvested embryos were frozen in the OCT, sectioned at 10μm thickness and transferred to PEN slides (Zeiss). Slides were dipped in 95% ethanol for 2 min to fix the samples and stained with crystal violet stain (3% in ethanol) on ice. This was followed by dipping in 70% ethanol for 30–40 sec to remove the OCT and dehydration in 100% ethanol for 2 min. The periocular mesenchymal tissue was micro-dissected using Laser capture microscope (Zeiss AxioObserver.Z1 inverted microscope). 500 pg of RNA was isolated from each sample, converted to cDNA and amplified using Nugen Ovation kit (Nugen) to obtain 2–3 μg cDNA, which was then converted to cDNA library for RNA-sequencing analysis at core facility in Columbia University. The RNAseq data is available at the GEO repository under accession number GSE103402.
5
RNA-Seq Analysis of CD4+ T Cells
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