Total RNA was extracted from skeletal muscle biopsies of eight women with PCOS and six healthy women, and from primary myotubes from five women with PCOS and six healthy women using the Qiagen AllPrep DNA/RNA/miRNA universal kit (Qiagen). Agilent RNA 600 Nano kit and Bioanalyzer instrument (Agilent Technologies) was used to assess the quality of the total RNA samples (500 ng). Sequencing libraries were prepared according to the TruSeq stranded total RNA with the Ribo-Zero Gold protocol (Illumina), as previously described (Hiam et al. 2019 (link)). Quantification of libraries was performed using the Qubit dsDNA HS assay kit (Invitrogen) to ensure optimum cluster densities. Quality control for base pair size and purity was assessed using an Agilent high-sensitivity DNA chip and Bioanalyzer instrument (Agilent Technologies). Each library was diluted to 1 nM before being pooled and sequenced on the NovaSeq6000 (Illumina).
Bioanalyzer instrument
Manufactured by Agilent Technologies
Sourced in United States, Germany
The Bioanalyzer instrument is a microfluidics-based platform for the analysis of DNA, RNA, proteins, and cells. It provides automated electrophoretic separation and detection of these biomolecules, enabling rapid and sensitive analysis.
Automatically generated - may contain errors
Lab products found in correlation
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (7 mentions)
Ampure xp bead, by Beckman Coulter (4 mentions)
Nanodrop one, by Thermo Fisher Scientific (4 mentions)
Rneasy plus mini kit, by Qiagen (3 mentions)
Hiseq 2000 platform, by Illumina (3 mentions)
Agilent dna high sensitivity chip, by Agilent Technologies (3 mentions)
Agilent rna 600 nano kit, by Agilent Technologies (3 mentions)
Nextseq 500, by Illumina (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
Nextseq 500 instrument, by Illumina (2 mentions)
Fragment analyzer, by Agilent Technologies (2 mentions)
Rna 6000 nano chip, by Agilent Technologies (2 mentions)
Trizol solution, by Thermo Fisher Scientific (2 mentions)
Mirneasy kit, by Qiagen (2 mentions)
Genechip mouse gene 2.1 st array, by Thermo Fisher Scientific (2 mentions)
E z n a total rna kit, by Omega Bio-Tek (2 mentions)
Nebnext ultra 2 dna library prep kit for protocol, by Illumina (2 mentions)
Nebnext multiplex oligos for dual index primers set 1, by Illumina (2 mentions)
Nextseq 500 sequencer, by Illumina (2 mentions)
53 protocols using bioanalyzer instrument
1
Transcriptomic Analysis of PCOS Muscle Biopsies
2
RNA Extraction and RNA-seq Library Prep
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Immediately following the 6 h treatment, total RNA was extracted using the Qiagen AllPrep DNA/RNA/miRNA universal kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The quality of total RNA samples (500 ng) was assessed using the Agilent RNA 6000 Nano kit and Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA). Sequencing libraries were prepared according to Illumina TruSeq stranded total RNA with the Ribo‐Zero Gold protocol (Illumina, San Diego, CA, USA) as previously described (Moreno‐Asso et al., 2022 ). Qubit dsDNA HS assay kit (Thermo Fisher Scientific) was used for quantification of libraries, and quality control for base pair size and purity was examined using an Agilent high‐sensitivity DNA chip and Bioanalyzer instrument (Agilent Technologies). Sequencing was performed on the NovaSeq 6000 (Illumina).
3
Illumina Deep Sequencing of Fish Immune Repertoire
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Libraries for Illumina deep sequencing were prepared as described (26 (link)). For cDNA barcoding, the primers used for second strand cDNA contained 15 random nt (Table S1 ). The location of the first Cμ primer in the Cμ2 domain restricts amplification of IgHm mRNA, since IgHδ contains a Cμ1 domain. The resulting ds cDNA was amplified by PCR to add the Illumina adaptor sequences and to integrate a fish-specific index or barcode. The following VH/C combinations were analyzed: VH4/Cμ (primer VH4.1), VH5/Cμ (primer VH5.1), VH8/Cμ (primer VH8.1), VH4/Cτ (primerVH4.1), and VH5/Cτ (primer VH5.4). Final PCR with Illumina adapters were purified using Agencourt AMPureXP beads (Beckman Coulter, Brea, CA). Library quality was assessed on a “DNA High Sensitivity” chip with a bioanalyzer instrument (Agilent Technologies, Santa Clara, CA). Equal amounts of libraries were pooled for multiplexing, and pools were sequenced in paired end 2 × 300 pb runs using a MiSeq instrument (Illumina) and the MiSeq Reagent Kit v3 (600 cycles) (Illumina) according to the manufacturer recommendations.
This consensus read sequencing approach based on the incorporation of a unique random barcode (UID) in each cDNA molecule produced at the reverse transcription step permits an accurate quantification of clonotype frequencies, and a better correction of PCR/sequencing errors (26 (link)).
This consensus read sequencing approach based on the incorporation of a unique random barcode (UID) in each cDNA molecule produced at the reverse transcription step permits an accurate quantification of clonotype frequencies, and a better correction of PCR/sequencing errors (26 (link)).
4
Gut Microbial Diversity in Mussels
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Mussels collected from sites 3 and 4 were chosen for 18S rDNA metabarcoding analysis due to the opposite characteristics of their habitats of origin. Individuals analyzed from site 3 were designated as MF (Mussel Farm) and from site 4 as WT. Nucleic acids were extracted from gut samples using PureLink microbiome DNA Purification kit (Invitrogen, Waltham, MA, USA) according to the manufacturer´s instructions. DNA concentration and purity were analyzed with an Infinite® 200 PRO Nanoquant (Tecan Group Ltd., Männedorf, Switzerland), Qubit 3.0 (Thermo Scientific, Waltham, MA, USA), and a Bioanalyzer Instrument (Agilent Technologies, Santa Clara, CA, USA) with GQN ≥7.
Sequencing was performed on Illumina Miseq (Illumina, San Diego, CA, USA) using Nextera XT v3 600 cycle kit at Fraunhofer Foundation (Santiago, Chile). Samples were amplified by dual-indexing Illumina fusion primers that targeted the 18S rDNA V4 region for eukaryotes [58 (link)]. The manufacturer’s recommended protocol was used to perform the sequencing reaction on Illumina Miseq platform. Sequence reads data were archived at NCBI Sequence Read Archive (SRA) with the BioProject number: PRJNA762938.
Sequencing was performed on Illumina Miseq (Illumina, San Diego, CA, USA) using Nextera XT v3 600 cycle kit at Fraunhofer Foundation (Santiago, Chile). Samples were amplified by dual-indexing Illumina fusion primers that targeted the 18S rDNA V4 region for eukaryotes [58 (link)]. The manufacturer’s recommended protocol was used to perform the sequencing reaction on Illumina Miseq platform. Sequence reads data were archived at NCBI Sequence Read Archive (SRA) with the BioProject number: PRJNA762938.
5
AsCpf1 CRISPR Targeting Protocol
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AsCpf1 targets were identified with Benchling software, and selected based only on the presence of a predicted PAM site without considerations of predicted efficiency. gRNAs were synthesized as custom RNA oligonucleotides with standard desalting (Integrated DNA Technologies), and resuspended in Picopure system purified water (Hydro Systems) at 500 or 1000 ng/µl. Two different gRNAs were synthesized for mouse pronuclear injections (unique target sequence for each is indicated in bold): Target A (5′-TAATTTCTACTCTTGTAGATATGATATCAACATCTACGACCTC -3′) and Target B (5′-TAATTTCTACTCTTGTAGATCTTGTTATTGTGGGAACAAGAAA -3′). The hAsCpf1 plasmid (Addgene 69982) was a gift from Feng Zhang. A PCR product amplified from the hAsCpf1 plasmid (Cpf1_Fwd actggcttatcgaaattaatacgactc-3′; Cpf1_Rev ccccagctggttctttcc-3′) was used as a template for in vitro RNA synthesis using mMessage mMachine T7 Ultra Transcription Kit (Thermo Fisher/Ambion), according to the manufacturer’s instructions. RNA was recovered using the MEGAclear Transcription Clean-up Kit (Thermo Fisher/Ambion). RNA quality was assessed with a Bioanalyzer instrument (Agilent Genomics), and stored in aliquots at −80° until used for injections.
6
Differential Gene Expression in Beta-Barr1-KO Mice
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Total RNA was extracted from isolated pancreatic islets of beta-barr1-KO and their control littermates that had been maintained on a HFD for ~10 weeks after the induction of the barr1 knockout. RNAs were tested for quality using a Bioanalyzer Instrument (Agilent, Santa Clara, CA). RIN >8 was the threshold used for determination of RNA quality. High-throughput sequencing was performed using a HiSeq 2500 Sequencing System (Illumina). The mouse genome mm9 was used to map the raw data. The Genomatix genome analyzer was employed to identify differentially expressed genes. Enrichment analysis and the analysis of biological pathways were performed using Metacore (Clarivate Analytics), Ingenuity Pathway Analysis (Qiagen), and Partek Flow (Partek). For the generation of heatmaps, functional gene enrichment analyses were performed using ToppGene Suite (http://toppgene.cchmc.org ). Genes enriched in selected GO pathways were visualized as heatmaps using Partek Flow. The RNA-seq data can be downloaded from the NCBI Sequence Read Archive under reference number RNA seq-PRJNA578926.
7
RNA Sequencing Library Preparation and Sequencing
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Total RNA was quality‐checked on the Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA) using the RNA 6000 Nano Chip (Agilent, Cat# 5067‐1511) and quantified by spectrophotometry using the NanoDrop ND‐1000 Instrument (NanoDrop Technologies, Wilmington, DE, USA). 1 μg total RNA was used for library preparation with the TruSeq Stranded mRNA Library Prep Kit High Throughput (Cat# RS‐122‐2103, Illumina, San Diego, CA, USA). Libraries were quality‐checked on the Fragment Analyzer (Advanced Analytical, Ames, IA, USA) using the Standard Sensitivity NGS Fragment Analysis Kit (Cat# DNF‐473, Advanced Analytical). The average concentration was 128 ± 12 nM. Samples were pooled to equal molarity. Each pool was quantified by PicoGreen fluorometric measurement to be adjusted to 1.8 pM and used for clustering on the NextSeq 500 instrument (Illumina). Samples were sequenced using the NextSeq 500 High Output Kit (75 cycles) (Illumina, Cat# FC‐404‐1005). Primary data analysis was performed with the Illumina RTA version 2.4.11 and base‐calling software version bcl2fastq‐2.20.0.422.
8
Quantifying Cellular RNA Synthesis
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After UVR cells were incubated with 1 mM 5-ethynyl-uridine (EU) for 1 h and subsequently fixated with 3.7% formaldehyde in PBS for 15 min. EU incorporated into newly synthesized RNA was detected using the Click-iT RNA Alexa Fluor 594 Imaging Kit (Invitrogen) following the manufacturer’s recommendations. Images were obtained using a fluorescence microscope (Leica DMIL). Fluorescence signal intensity was measured in ≥100 nuclei and expressed in arbitrary units using ImageJ software. Total, mRNA and rRNA were measured in triplicates by spectrophotometry (NanoDrop) and fluorescence electropherogram on a Bioanalyzer instrument (Agilent) and normalized to cell numbers. To determine RNA content per cell, cells were stained using the metachromatic dye acridine orange (AO, Sigma Aldrich) at a concentration of 10 μg/ml and subsequently analyzed by flow cytometry on a BD FACS Canto.
9
Quantitative RT-qPCR Analysis of Gene Expression
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Total RNAs were extracted from cell samples using TRIzol solution (Invitrogen). The quality of RNAs was assessed using a Bioanalyzer instrument (Agilent) and then quantified using a Biospecnano (Shimadzu, Kyoto, Japan). cDNA synthesis was prepared from 1 μg of total RNA with random hexamers using Applied Biosytems Reverse Transcription kit according to the supplied protocols. Gene expression was quantified by SYBR Green qPCR method using the Maxima™ SYBR Green/ ROX qPCR Master Mix on an StepOnePlus Real Time PCR system (ThermoFisher Scientific). Relative expression was calculated by using the comparative Ct method (2-ΔΔCt). Primer sequences for the quantification of 35 genes were purchased from Sigma and are available upon request. Transcript levels of HPRT for PBMC, or 18S for melanoma cells lines were used as endogenous control.
10
RNA Extraction and Sequencing Workflow
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RNA extraction was undertaken using the RNeasy Plus Micro Kit and RNeasy Plus Mini Kit (Qiagen). For each sample collected, the concentration of purified RNA was measured using the Qubit™ RNA HS Assay Kit (Thermofisher). RNA was pooled to reach a concentration of 2–4 μg of RNA. A total of 10 cases and 5 control RNA sequencing libraries were prepared. All RNA sequencing libraries included samples from both nasal and nasopharyngeal swabs. The RNA quality of the pools was measured using a Bioanalyzer instrument (Agilent). Library preparation was completed using TruSeq Stranded Total RNA Human/Mouse/Rat Kit (Illumina) that removes cytoplasmic rRNA and sequenced in an Illumina NovaSeq S1 platform (paired-end sequencing with 150 cycles per read) at the Australian Genome Research Facility (AGRF).
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