DNA was extracted from the swabs using a DNA extraction kit (IngeniGen XMK Biotechnologies Inc., Zhejiang, China), according to the manufacturer’s instructions. Before DNA extraction, an internal control bacterium was added to the samples. DNA concentration was measured using a Qubit® 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA). DNA libraries were constructed using the Ingenigen XMKbio DNA-seq Library Prep Kit (IngeniGen XMK Biotechnologies, Inc.) using the Tn5 transposase method. DNA library concentrations were measured using a Qubit® 4.0 Fluorometer (Thermo Fisher Scientific), and their quality was evaluated using an Agilent 4200 TapeStation system (Agilent Technologies, Santa Clara, CA). Qualified libraries with different barcodes were then pooled accordingly. Blank tubes with unused swabs and sterile water were used as blank extraction-negative controls during DNA extraction and library preparation to filter reagent and laboratory environmental contamination taxa. The “environmental” species with a frequency of more than 10% in the negative controls (pre-determined by ingeniSeq-MG V1.0 mNGS software) over the past 100 runs were considered as contaminants and filtered out from the final results. Sequencing was performed on an Illumina Nextseq550 using a 75-bp single-end sequencing mode.
Qubit 4.0 fluorometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Singapore, Italy, Germany
The Qubit 4.0 fluorometer is a compact and precise instrument used for quantifying nucleic acids and proteins. It utilizes fluorescent dyes to measure the concentration of these biomolecules in a sample with high sensitivity and accuracy.
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Lab products found in correlation
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (86 mentions)
Novaseq 6000, by Illumina (16 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (14 mentions)
Nanodrop, by Thermo Fisher Scientific (14 mentions)
Novaseq 6000 platform, by Illumina (13 mentions)
Agencourt ampure xp bead, by Beckman Coulter (13 mentions)
Qiaamp dna mini kit, by Qiagen (11 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (11 mentions)
Tguide s96 magnetic soil stool dna kit, by Tiangen Biotech (11 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (9 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (9 mentions)
Magmax cell free dna isolation kit, by Thermo Fisher Scientific (8 mentions)
Dneasy blood and tissue kit, by Qiagen (8 mentions)
Qubit 1x dsdna hs assay kit, by Thermo Fisher Scientific (8 mentions)
Qubit rna br assay kit, by Thermo Fisher Scientific (7 mentions)
Qiaamp circulating nucleic acid kit, by Qiagen (7 mentions)
Miseq platform, by Illumina (7 mentions)
Tapestation 4150, by Agilent Technologies (7 mentions)
Agilent 4200 tapestation system, by Agilent Technologies (6 mentions)
Dsdna hs assay kit, by Thermo Fisher Scientific (6 mentions)
307 protocols using qubit 4.0 fluorometer
1
Microbiome DNA Extraction and Sequencing
2
Fecal Microbiome Profiling via 16S rRNA Sequencing
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Fecal genomic DNA was extracted using the TGuide S96 Magnetic Stool DNA Kit (Tiangen Biotech Co., Ltd. Beijing, China), according to manufacturer’s instructions. The DNA quality was assessed using the Qubit dsDNA HS Assay Kit and Qubit 4.0 Fluorometer (Invitrogen, Oregon, United States). The special region (V3-V4) of the 16S rRNA gene in DNA samples was amplified using the general primers 338FP 5′-ACTCCTACGGGAGGCAGCA-3′ and 806RP 5′-GGACTACHVGGGTWTCTAAT-3'. The reaction volume for the polymerase chain reaction (PCR) was 10 μl, including 338FP (10 μM) 0.3 μl, 806RP (10 μM) 0.3 μl, KOD FX Neo 0.2 μl, KOD FX Neo Buffer 5 μl, dNTP (2 μM) 2 μl, DNA template 25 ng, and distilled water up to 10 μl. The PCR products were further purified using Agencourt AMPure XP Beads (Beckman Coulter, Indianapolis, IN, United States) and quantified using the Qubit 4.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, Oregon, United States). Then, the purified PCR products were mixed in equal amounts to construct a library. The library was sequenced on Illumina Novaseq 6000, and further bioinformatic analysis was carried out using BMKCloud (Biomarker Technologies Co., Ltd. Beijing, China).
3
Isolation of NAc RNA from METH-Exposed Rats
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Rats were anaesthetized using Zoletil 50 (1 mL/kg, intraperitoneal) and rapidly decapitated. There were 8 METH self-administered rats and 6 saline controls. Brain tissues were dissected and isolated and then sliced using a rat brain matrix (2.2–0.8 mm according to coordinates from the Paxinos and Watson (1998) rat brain atlas). The NAc was harvested bilaterally using a stainless-steel cannula. Total RNA was collected using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The integrity and quantity of the RNA sample were assessed using a Qubit 4.0 fluorometer (Invitrogen, Life Technologies). High-quality samples were most important for the small RNA sequencing projects. The amount of purified total RNA was approximately 1–2 µg. The RNA integrity (RIN) needed to be greater than 6.5, as detected by a Qubit 4.0 fluorometer (Invitrogen, Life Technologies).
4
RNA-Seq Library Preparation Protocol
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RNA samples were quantified using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA, USA), and RNA integrity was checked with an RNA Kit on an Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following the manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94°C. First-strand and second-strand cDNAs were subsequently synthesized. cDNA fragments were end-repaired and adenylated at 3’ ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment via limited-cycle PCR. Sequencing libraries were validated using the NGS Kit on the Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified with the Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).
5
Illumina-Based Soil Microbiome Analysis
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The DNA was extracted with the TGuide S96 Magnetic Soil /Stool DNA Kit (Tiangen Biotech (Beijing) Co., Ltd.) according to manufacturer instructions. The DNA concentration of the samples was measured with the Qubit dsDNA HS Assay Kit and Qubit 4.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, Oregon, United States). The total of PCR amplicons was purified with Agencourt AMPure XP Beads (Beckman Coulter, Indianapolis, IN) and quantified using the Qubit dsDNA HS Assay Kit and Qubit 4.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, Oregon, United States). After the individual quantification step, amplicons were pooled in equal amounts. For the constructed library, use Illumina novaseq 6,000 (Illumina, Santiago CA, United States) for sequencing. The original sequence was processed by BMK Cloud (Biomarker Technologies Co., Ltd., Beijing, China) and was used as a marker for SSU rRNA classification with the SILVA database. The phylogenetic relationship among the bacterial communities was analyzed, and the determination of bacterial alpha diversity, construction of principal coordinate analysis (PCoA) map, and functional prediction of the bacterial communities were then performed using the QIIME2.1
6
Microbial DNA and RNA Extraction and Sequencing
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Biomass sampling was performed when switching incubation pressure (Fig. 1C ). Total microbial genomic DNA was extracted and purified using the modified SDS-based method described by Natarajan et al.43 (link), and stored at −20 °C before further assessment. The quantity and quality of extracted DNA were measured using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and agarose gel electrophoresis, respectively. The extracted microbial DNA was processed to construct metagenome shotgun sequencing libraries with insert sizes of 350 bp following the standard Illumina TruSeq DNA Sample Preparation Guide. Each library was sequenced by Illumina NovaSeq 6000 platform (Illumina, USA) with PE150 strategy at Shanghai Personal Biotechnology (Shanghai, China). The extraction of RNA from sediment samples was carried out using the RNeasy® PowerSoil® Total RNA Kit (Cat. No. 12866-25, Qiagen, Germany) according to the manufacturer’s instructions, then quantified using a Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). To ensure DNA removal, the RNA extracts were treated with TURBO DNase (Cat. No. AM2238, Invitrogen, Waltham, MA, USA) as directed by the manufacturer. The purified RNA was converted to cDNA, then the metatranscriptomic library was constructed by using Illumina TruSeq Stranded mRNA LT Sample Prep Kit, and subsequent sequencing as described above.
7
Metagenomic Profiling of Soil/Stool Samples
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The DNA was extracted with the TGuide S96 Magnetic Soil/Stool DNA Kit (Tiangen Biotech (Beijing) Co., Ltd.) according to manufacturer’s instructions. The DNA concentration of the samples was measured with the Qubit dsDNA HS Assay Kit and Qubit 4.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, Oregon, United States). The 338F: 5′-ACTCCTACGGGAGGCAGCA-3′ and 806R: 5′-GGACTACHVGGGTWTCTAAT-3′ universal primer set was used to amplify the V3-V4 region of the 16S rRNA gene. The ITS1F: 5′-CTTGGTCATTTAGAGGAAGTAA-3′ and ITS2: 5′-GCTGCGTTCTTCATCGATGC-3′ universal primer set was used to amplify the ITS1 region of the ITS gene. Both the forward and reverse primers were tailed with sample-specific Illumina index sequences to allow for deep sequencing. The total PCR amplicons were purified with Agencourt AMPure XP Beads (Beckman Coulter, Indianapolis, IN) and quantified using the Qubit dsDNA HS Assay Kit and Qubit 4.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, Oregon, United States). After the individual quantification step, amplicons were pooled in equal amounts. For the constructed library, an Illumina NovaSeq 6000 (Illumina, Santiago CA, United States) was used for sequencing.
8
RNA Isolation and Integrity Assessment
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Total RNA was isolated using mirVana miRNA Isolation Kit (Ambion) following the manufacturer’s instruction. RNA purity was evaluated with a Nanodrop ND-1000 spectrophotometer (v.3.5.2, Thermo Fisher Scientific). QubitTM 4.0 Fluorometer with the Qubit RNA BR (broad-range) assay kit (Thermo Fisher Scientific) was used to quantify RNA concentration. The assessment of RNA integrity was performed using Agilent 2100 Bioanalyser (Agilent Technologies). Only RNA with integrity number greater than 8.0 was used for library preparation for RNA-seq, CAGE-seq, and ISO-seq.
9
Chloroplast DNA Extraction and Sequencing
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The washed chloroplasts pellet was lysed by suspension in 500 µL of AP1 buffer with 5 µL of RNAse A (100 mg/mL) (DNeasy Plant Mini Kit (Cat. No. 69106), QIAGEN, Hilden, Germany) and incubated at 65 °C in a water bath for 50 min. Once the suspension turned a dark olive green color, the tubes were removed. From this point onward, cpDNA was extracted using DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) and eluted to a final volume of 70 µL according to the manufacturer’s protocol. Eight microliters of cpDNA were checked for quality by gel electrophoresis on a 1.2% agarose gel in 1x TAE buffer (Figure 2 ). The cpDNA concentration was measured using a 1x dsDNA HS Assay kit of QubitTM 4.0 Fluorometer (Thermo Fisher Scientific, Singapore) for each sample, to determine cpDNA yield prior to Illumina MiSeq (2 × 300 bp) paired-end sequencing.
10
Amplicon Library Preparation for MiSeq
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Amplicon DNA concentrations were quantified using the Qubit4.0 Fluorometer (Life Technologies, Stockholm, Sweden). Amplicons were combined in equimolar ratios into a single tube with a final concentration of the DNA library of 4 pM. As an internal control, 5% of PhiX was added to the amplicon pool. Paired-end sequencing with a read length of 2 × 250 bp was carried out on a Miseq Instrument (Illumina) using a Miseq reagent kit v2 (Illumina).
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