For 16S rRNA sequencing and transcriptome sequencing, microbial DNA and mRNA were extracted from specific tissues or cells of interest at a selected time point. Illumina platform-based sequencing was carried out using the NovaSeq Reagent Kit method for library construction, and bioinformatics analysis was performed using advanced computational tools to analyze the sequencing data. For eukaryotic mRNA sequencing, Illumina sequencing technology was employed to sequence all transcripts of interest in a specific tissue or cell at a certain time point. The sequencing experiment was performed using the Illumina NovaSeq Reagent Kit method for library construction, and analysis of the sequencing data was carried out using a bioinformatics platform. The raw data was deposited in the NCBI BioProject database (BioProject ID: PRJNA981653).
Novaseq reagent kit
Manufactured by Illumina
Sourced in China, United States
The NovaSeq Reagent Kits are a set of consumables designed for use with the NovaSeq 6000 Sequencing System, a high-throughput DNA sequencing platform manufactured by Illumina. The kits contain the necessary reagents and materials required to perform DNA sequencing experiments on the NovaSeq 6000 system.
Automatically generated - may contain errors
Lab products found in correlation
Nextflex rapid dna seq, by PSC Biotech (13 mentions)
E z n a soil dna kit, by Omega Bio-Tek (9 mentions)
Novaseq, by Illumina (9 mentions)
Novaseq 6000, by Illumina (9 mentions)
Hiseq x reagent kit, by Illumina (7 mentions)
Kapa library quantification kit, by Roche (5 mentions)
Novaseq hiseq xten, by Illumina (5 mentions)
Fragment analyzer, by Agilent Technologies (5 mentions)
Novaseq platform, by Illumina (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Nextflextm rapid dna seq, by PSC Biotech (3 mentions)
Novaseq s4 lane, by Illumina (3 mentions)
Truseq nano dna ht library prep kit, by Illumina (2 mentions)
M220 focused ultrasonicator, by Covaris (2 mentions)
Fastdna spin kit for soil, by MP Biomedicals (2 mentions)
Fragment analyzer, by Roche (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Nextflex small rna seq kit v3, by PSC Biotech (2 mentions)
Truseq mrna, by Illumina (1 mentions)
41 protocols using novaseq reagent kit
1
Comprehensive Multi-Omics Microbial and Eukaryotic Profiling
2
Illumina Sequencing of DNA Fragments
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DNA extract was fragmented to an average size of about 400 bp using Covaris M220 (Gene Company Limited, China) for paired-end library construction. Paired-end library was constructed using NEXTFLEX® Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Adapters containing the full complement of sequencing primer hybridization sites were ligated to the blunt-end of fragments. Paired-end sequencing was performed on Illumina NovaSeq (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits according to the manufacturer’s instructions (www.illumina.com ). The data were analyzed on the free online platform of Majorbio Cloud Platform (www.majorbio.com ). The paired-end Illumina reads were trimmed of adaptors, and low-quality reads (length<50 bp or with a quality value<20 or having N bases) were removed by FASTp (https://github.com/OpenGene/fastp , version 0.20.0).
3
Genomic DNA Extraction and Sequencing of A. ruthenus
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The genomic DNA of 31 female and 30 male A. ruthenus specimens was extracted from 90% ethanol-preserved fin clips using a classical phenol/chloroform protocol, quantified using Qubit fluorimetry and analysed using the Fragment Analyzer (Advanced Analytical Technologies, Inc., Iowa, USA). DNA short read sequencing was performed at the GeT-PlaGe core facility, INRAe Toulouse (https://get.genotoul.fr/en/ ). Two DNA pool-seq libraries were prepared according to manufacturer's protocols using the Illumina TruSeq Nano DNA HT Library Prep Kit (Illumina, California, USA). Briefly, from 200 ng of each sample, DNA was fragmented (550 bp) by sonication on a M220 Focused-ultrasonicator (COVARIS). Size selection was performed using SPB beads (kit beads) and 3′-ends of the blunt fragments were mono-adenylated. Then, adaptors and indexes were ligated and the construction amplified with Illumina-specific primers (eight cycles). Library-quality was assessed using Fragment Analyzer and libraries were quantified by qPCR using the Kapa Library Quantification Kit (Roche). Sequencing was performed on a NovaSeq S4 lane (Illumina, California, USA), using a paired-end read length of 2 × 150 bp with the Illumina NovaSeq Reagent Kits.
4
Soil Metagenomic DNA Extraction and Sequencing
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Three soil cores from the upper 10 cm were randomly sampled at the end of each extreme drought event from each plot, soil cores from the same plot were mixed, homogenized, sieved through a 2.0 mm mesh size sieve, and immediately stored at −20 °C prior to DNA extraction. Total genomic DNA was extracted from soil samples using the E.Z.N.A. Soil DNA Kit (Omega Bio-Tek, Norcross, GA, U.S.). The concentration and purity of extracted DNA were determined by TBS-380 and NanoDrop2000 respectively. The quality of the DNA extract was determined by 1% agarose gel.
DNA extracts were segmented to an average size of about 400 bp using Covaris M220 (Gene Company Limited, China) for the construction of paired terminal libraries. NEXTFLEX Rapid DNA-Seq was used to construct the paired-end library (Bioo Scientific, Austin, TX, USA). Paired-end sequencing was performed on Illumina NovaSeq 6000 (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits (www.illumina.com ).
DNA extracts were segmented to an average size of about 400 bp using Covaris M220 (Gene Company Limited, China) for the construction of paired terminal libraries. NEXTFLEX Rapid DNA-Seq was used to construct the paired-end library (Bioo Scientific, Austin, TX, USA). Paired-end sequencing was performed on Illumina NovaSeq 6000 (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits (
5
RNA-seq analysis of neurodegenerative models
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For the RNA-seq of the mouse samples, total RNA was extracted from MACS-isolated microglia of each models using an RNeasy Mini Kit (Qiagen, Hilden, Germany). The RNAs were sampled from cerebral cortices of 8-month-old AppNL-G-F/NL-G-F and 7-month-old rTg4510 mice and lumbar spinal cords of 5-month-old SODG93A mice together with the corresponding wild-type or control mice, respectively. For the RNA-seq of the human samples, total RNA was prepared from precuneus of frozen postmortem brain using mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer instructions. The total RNA was qualified by using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Libraries were prepared by using TruSeq mRNA or TruSeq Stranded mRNA (Illumina, San Diego, CA, USA), and, from these libraries, 151-nt paired-end reads were sequenced on the HiSeq X Ten with the HiSeq X Reagent Kits (Illumina) and the NovaSeq 6000 with the NovaSeq Reagent Kits (Illumina).
6
Cecal Microbiota Profiling by Metagenomics
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Metagenome sequencing was used to investigate the cecal microbiota in greater detail, as previously described (41 (link)), with some modifications. To summarize, total genomic DNA was extracted from the cecal contents 26 of chickens SE-infected using the E.Z.N.A.® Soil DNA Kit (Omega Bio-Tek, Norcross, GA, United States) according to the manufacturer’s protocol. TBS-380 and NanoDrop2000 were used to determine the concentration and purity of extracted DNA, respectively. On a 1% agarose gel, the DNA quality was determined. The extracted DNA was then fragmented to an average size of approximately 400 bp using the Covaris M220 (Gene Company Limited, China) to construct paired-end libraries. Using NEXTflexTM Rapid DNA-Seq, a paired-end library was built (Bioo Scientific, Austin, TX, USA). To the blunt end of the fragments, adapters containing the complete complement of sequencing primer hybridization sites were ligated. On the Illumina NovaSeq/Hiseq Xten, paired-end sequencing was performed (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits/HiSeq X Reagent Kits according to the manufacturer’s instructions (www.illumina.com ).
7
Gut Microbiome DNA Extraction and Sequencing
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Total genomic DNA was extracted from colon content samples using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. The concentration and purity of extracted DNA were determined with TBS-380 and NanoDrop2000, respectively. DNA extract quality was checked on 1% agarose gel.
DNA extract was fragmented to an average size of about 400 bp using Covaris M220 (Gene Company Limited, Hong Kong, China) for paired-end library construction. Paired-end library was constructed using NEXTFLEX Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Adapters containing the full complement of sequencing primer hybridization sites were ligated to the blunt end of fragments. Paired-end sequencing was performed on Illumina NovaSeq/Hiseq Xten (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits/HiSeq X Reagent Kits according to the manufacturer’s instructions (www.illumina.com , accessed on 10 December 2021). Sequence data associated with this project were deposited in the NCBI Short Read Archive database (Accession Number: PRJNA849732).
DNA extract was fragmented to an average size of about 400 bp using Covaris M220 (Gene Company Limited, Hong Kong, China) for paired-end library construction. Paired-end library was constructed using NEXTFLEX Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Adapters containing the full complement of sequencing primer hybridization sites were ligated to the blunt end of fragments. Paired-end sequencing was performed on Illumina NovaSeq/Hiseq Xten (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using NovaSeq Reagent Kits/HiSeq X Reagent Kits according to the manufacturer’s instructions (
8
Illumina-based Whole Genome Sequencing
9
Gut Microbiome DNA Sequencing and Analysis
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Total DNA was extracted from 1 g of faeces using a kit (Omega Bio-Tek, Norcross, GA, United States), and the concentration, purity and quality of the DNA was determined[15 (link)].
The extracted DNA (average length of 400 bp) was fragmented using a Covaris M220 ultrasonicator (Gene Company Limited, China). A paired-end library was constructed using NEXTFLEX Rapid DNA-Seq (Bioo Scientific, Austin, TX, United States). Sequencing was performed with an Illumina NovaSeq system (Illumina Inc., San Diego, CA, United States) using NovaSeq Reagent Kits according to the manufacturer’s instructions[16 (link)]. The paired-end Illumina reads were trimmed of adaptors, and reads with low quality (length < 50 bp, quality value < 20, containing N bases) were removed by fastp[17 (link)].
Metagenomic data were collected using MEGAHIT. Contigs with a length ≥ 300 bp were selected as the final assembly output and used for further gene annotation. Amino acid sequences from the predicted open reading frames with a length ≥ 100 bp were retrieved and translated from the NCBI database[18 (link)]. The nonredundant gene catalogue was constructed using CD-HIT and aligned to high-quality reads using SOAP aligner.
The extracted DNA (average length of 400 bp) was fragmented using a Covaris M220 ultrasonicator (Gene Company Limited, China). A paired-end library was constructed using NEXTFLEX Rapid DNA-Seq (Bioo Scientific, Austin, TX, United States). Sequencing was performed with an Illumina NovaSeq system (Illumina Inc., San Diego, CA, United States) using NovaSeq Reagent Kits according to the manufacturer’s instructions[16 (link)]. The paired-end Illumina reads were trimmed of adaptors, and reads with low quality (length < 50 bp, quality value < 20, containing N bases) were removed by fastp[17 (link)].
Metagenomic data were collected using MEGAHIT. Contigs with a length ≥ 300 bp were selected as the final assembly output and used for further gene annotation. Amino acid sequences from the predicted open reading frames with a length ≥ 100 bp were retrieved and translated from the NCBI database[18 (link)]. The nonredundant gene catalogue was constructed using CD-HIT and aligned to high-quality reads using SOAP aligner.
10
Fecal Metagenomic DNA Sequencing
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Total genomic DNA was extracted from fecal samples using the E.Z.N.A.® Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA). DNA concentration and purity were determined using Qubit4.0 and NanoDrop2000, respectively. Genomic DNA was fragmented to an average size of about 400 bp using Covaris M220 (Gene Company Limited, China) for paired-end library construction. The NEXTflex™ Rapid DNA-Seq Kit (Bioo Scientific, Austin, TX, USA) was used to build the paired-end library. The blunt ends of fragments were ligated with adapters containing a full complement of hybridization sites for sequencing primers. Shotgun sequencing was performed on the Illumina NovaSeq (Illumina Inc., San Diego, CA, USA) at Honsunbio Technology Co., Ltd. (Shanghai, China) with NovaSeq Reagent Kits (www.illumina.com ).
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