RNA was extracted using Qiagen Allprep DNA/RNA/miRNA universal kit, which simultaneously isolates genomic DNA and total RNA. RNA concentration was evaluated using a NanoDrop spectrophotometer (ThermoFisher, Waltham, Massachusetts, USA) and RNA integrity was evaluated using the Bioanalyzer RNA 6000 Nano kit (Agilent Technologies, Santa Clara, California, USA). MiRNA NGS libraries were then prepared using TruSeq Small RNA Library protocol and sequences using HiSeq 2500 High Throughput Sequencer (all from Illumina, San Diego, California, USA).
Bioanalyzer rna 6000 nano kit
Manufactured by Agilent Technologies
Sourced in United States
The Bioanalyzer RNA 6000 Nano Kit is a laboratory equipment product designed for the analysis of RNA samples. It provides a sensitive and accurate assessment of RNA quality and quantity using microfluidic technology.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (12 mentions)
Nanodrop, by Thermo Fisher Scientific (10 mentions)
Nextseq 500, by Illumina (6 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Qiacube, by Qiagen (3 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (3 mentions)
Allprep dna rna mirna universal kit, by Qiagen (2 mentions)
Truseq stranded mrna kit, by Illumina (2 mentions)
Nextseq 500 platform, by Illumina (2 mentions)
Mirneasy kit, by Qiagen (2 mentions)
Qbase software, by CellCarta (2 mentions)
Rneasy minelute cleanup kit, by Qiagen (2 mentions)
Maxwell nucleic acid extraction instrument, by Promega (2 mentions)
Maxwell 16 lev simplyrna cells kit, by Promega (2 mentions)
Paxgene blood mirna kit, by Qiagen (2 mentions)
Tri reagent, by Merck Group (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480, by Roche (2 mentions)
Bioanalyzer rna 6000 pico kit, by Agilent Technologies (2 mentions)
51 protocols using bioanalyzer rna 6000 nano kit
1
Comprehensive miRNA Sequencing Protocol
2
Transcriptome Analysis of Gene Silencing
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RNA was quantified by Nanodrop (Thermo Scientific, Waltham, Massachusetts, USA) and quality assessment was performed by Bioanalyzer -RNA 6000 nano kit (Agilent Technologies, Santa Clara, California, USA).
Libraries were prepared starting from 1 μg RNA using TruSeq Stranded mRNA kit (Illumina, San Diego, California, USA). Next generation sequencing was conducted on NextSeq 500 platform (Illumina, San Diego, California, USA) and a minimum of 30 million of reads for each replicate was expected. Cufflink RNA-Seq workflow was applied to perform bioinformatic analysis. Differential gene expression was calculated as log2 fold-change (siRUNX2/siCtrl, siHDAC6/siCtrl). Differential expression p-values were adjusted by an optimized FDR approach (FDR cutoff = 0.05) and genes with adjusted p-value (q-value) < 0.05 were considered significantly deregulated.
Libraries were prepared starting from 1 μg RNA using TruSeq Stranded mRNA kit (Illumina, San Diego, California, USA). Next generation sequencing was conducted on NextSeq 500 platform (Illumina, San Diego, California, USA) and a minimum of 30 million of reads for each replicate was expected. Cufflink RNA-Seq workflow was applied to perform bioinformatic analysis. Differential gene expression was calculated as log2 fold-change (siRUNX2/siCtrl, siHDAC6/siCtrl). Differential expression p-values were adjusted by an optimized FDR approach (FDR cutoff = 0.05) and genes with adjusted p-value (q-value) < 0.05 were considered significantly deregulated.
3
RNA Isolation and Sequencing Protocol
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Tissues were homogenized into fine powders in liquid nitrogen using a mortar and pestle. RNAs were extracted using miRNeasy kit (Qiagen, US) following manufacturer’s protocol. Quality of extracted RNAs were assessed using Bioanalyzer RNA 6000 nano kit (Agilent Technologies, US). RNA samples with RIN value > = 8 were pursued for RNA quantification using Qbit (Thermo Fisher, US). RNA-sequencing library preparation was done using Illumina TruSseq ribo-zero gold kit following manufacturer’s instructions. For each sample, 1 μg of total RNA was used for sequencing library preparation. After libraries were prepared for each sample, quantification of library was performed using Kapa quantification kit (Kapa systems) using ABI7300 instrument. Libraries were further normalized to ensure equal quantity before sequencing. 2 × 150 bp paired-end reads were obtained using Illumina NextSeq 500 instrument with high-output kit.
4
Bulk RNA Sequencing of Regulatory T Cells
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FACS-sorted tTreg from WT-Foxp3RFP and Kcnk18G339R/Foxp3RFP thymus were used for bulk RNA sequencing. RNA was isolated with Quick-RNA Microprep Kit (Zymo Research) as described above. Quality and amount of RNA were verified by NanoDrop and Bioanalyzer RNA 6000 Nano Kit (Agilent). Samples with RIN values > 6.5 were used for RNA sequencing. NEBNext® rRNA depletion was performed followed by NEBNext directional Ultra RNA II Library preparation and sequencing on NextSeq500 (Illumina) platform (75 cycles, high output v2 kit). Raw sequencing data were analyzed by Linux bash tools following the analysis pipeline: (1) quality control (fastqc), (2) trimming (Trimmomatic 0.36),61 (link) (3) alignment to mouse genome (Hisat 2.1.0; build: mm10),62 (link) (4) aligned read sorting (Samtools)63 (link) and (5) read counting (HTseq 0.10.0.64 (link)) Expression analysis was performed with R/Bioconducter DESeq2.65 (link) Treg signature genes were assigned according to a previous study.66 (link) Significantly regulated genes (FDR < 0.05) were used for further analysis and visualization using pHeatmap (https://cran.r-project.org/package=pheatmap ) package in R. GO term gene enrichment analysis was performed using the PANTHER classification system online tool.67 (link),68 (link)
5
Quantitative Analysis of Bone Marrow Gene Expression
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Whole, marrow flushed femora were collected and homogenised using the Omni tissue homogeniser (Omni, Kennesaw, GA, USA) in Trizol reagent (Ambion, Austin, TX, USA). RNA was extracted using the NucleoSpin RNA Plus extraction kit (Macherey-Nagel, Düren, Germany). RNA quality was confirmed with the Qubit RNA HS assay kit (Invitrogen, Carlsbad, CA, USA) and the Bioanalyzer RNA 6000 Nano kit (Agilent, Santa Clara, CA, USA). For qPCR analysis, RNA was primed with random primers and OligoDT and cDNA was prepared using the SuperScript III First-Strand Synthesis System (ThermoFisher Scientific). PCR was carried out on the LightCycler 480 (Roche, Basel, Switzerland) using the SYBR Green master mix (Roche). Gene expression was quantified relative to three housekeeping genes using the LightCycler 480 software (Roche) and the qBase+ software (Biogazelle, Ghent, Belgium).
6
Post-mortem Brain Tissue Preservation and Analysis
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As per data source, fresh post-mortem brain (only specimens from neurologically healthy individuals that were free from significant genetic errors) were considered for original data retrieval. In addition, to ensure consistency between the samples and to decrease potential variation arising from ante- and post-mortem conditions, specific tissue preservation, storage, and RNA extraction and analysis criteria were followed (all described in detail at https://help.brain-map.org/display/devhumanbrain/Documentation ). Briefly, RNA quality was confirmed using Bioanalyzer RNA 6000 Nano Kit or Bioanalyzer RNA 6000 Pico Kit (Agilent), RNA sequencing was performed using an Illumina Genome Analyzer II (GAIIx) instrument, and Gencode (v10 and Gencode v3c annotations) was used for RNA sequencing alignment and expression quantification. The metadata for the subjects included in this study were age, gender, ethnicity, and post-mortem interval (PMI), pH, cerebral hemisphere used for the tissue biopsy, and RNA integration number (RIN); the quality of the data are given in Table S1. All work was performed according to guidelines for the research use of human brain tissue and with prior approval by the Human Investigation Committees and Institutional Ethics Committees of each institute from which samples were obtained.
7
RNA-Seq analysis of activated pathways
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Total RNA was isolated using Isogen II (NipponGene) according to the manufacturer’s protocol. The RNA integrity number of the purified RNAs was evaluated using the Bioanalyzer RNA6000 Nano kit (Agilent Technologies). In particular, 300 ng RNA was subjected to RNA sequencing using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina and NEBNext Poly(A) messenger RNA isolation kit (New England Bio Labs). Generated libraries were sequenced using the MiSeq Reagent Kit version 3 150-cycle kit on a MiSeq system (Illumina). Fragments per kilobase of exon per million fragments mapped read data were used for relative expression analysis. Visualizations of activated and inactivated signal pathways on the basis of the enriched genes were performed using Ingenuity Pathway Analysis software (Qiagen).
8
RNA-seq Analysis of Frozen Samples
9
Robust RNA Extraction from Rabbit Lungs
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After collection, lungs were immediately placed in RNA Later (Sigma Aldrich, USA) and stored at − 20 °C. Samples were homogenized in QIAzol® Lysis Reagent. Total RNA was extracted with the miRNeasy Mini Kit protocol (QIAGEN, Germany), using an automated method (QIAcube: QIAGEN, Germany) adapted to include DNase I treatment. RNA concentration was measured using Qubit 4 fluorometer (Thermo Fisher Scientific, USA). RNA integrity was assessed by checking the 2:1 ratio of 18S and 28S ribosomal RNA bands and RNA Integrity Number (RIN) by agarose gel electrophoresis and Bioanalyzer RNA 6000 Nano Kit (Agilent, USA), respectively. Lung-derived RNA was suitable for RNA-sequencing (RIN > 8). Libraries for high-throughput RNA sequencing were prepared using the QuantSeq FWD kit (Lexogen, Austria) and sequenced in three different runs with the Illumina NexSeq500 platform, which generated at least 20 million reads for each sample. 96% of the reads were mapped to the rabbit genome.
10
Quantifying Collagen Precursor Transcripts
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