Total RNA was extracted from Granta-519 cells infected with shctrl or shKIAA0101 lentiviruses. Next, the content and quality of RNA were analyzed using the Nanodrop 2000 (Thermo Fisher Scientific) and the Agilent 2100 Bioanalyzer (Agilent, Pal Alto, CA, USA). RNA (1.7< A260/A280 <2.2, RNA integrity number ≥7.0 and 28S/18S >0.7) was reverse transcribed into first-strand cDNA, which was further converted into double-stranded DNA and biotin-labeled amplified RNA (aRNA) using the GeneChip 3' IVT Express Kit (Affymetrix, Santa Clara, CA, USA). After purification, aRNA was quantified using the Nanodrop 2000 (Thermo Fisher Scientific), fragmented, and hybridized with the GeneChip Human Genome U133 plus 2.0 Array (Affymetrix). Next, hybridized chips were stained and washed using the GeneChip Hybridization Wash and Stain Kit (Affymetrix) on the Genechip Fluidics Station 450 instrument (Affymetrix). Finally, microarray signals were scanned and analyzed using the Genechip Array Scanner 3000 7G (Affymetrix).
Nanodrop 2000
Manufactured by Thermo Fisher Scientific
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The NanoDrop 2000 is a spectrophotometer designed for the analysis of small volume samples. It enables rapid and accurate quantification of proteins, DNA, and RNA by measuring the absorbance of the sample. The NanoDrop 2000 utilizes a patented sample retention system that requires only 1-2 microliters of sample for each measurement.
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Rneasy mini kit, by Qiagen (925 mentions)
Primescript rt reagent kit, by Takara Bio (609 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (572 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (470 mentions)
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Steponeplus real time pcr system, by Thermo Fisher Scientific (292 mentions)
Iscript cdna synthesis kit, by Bio-Rad (282 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (256 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (255 mentions)
Rnaiso plus, by Takara Bio (248 mentions)
Mirneasy mini kit, by Qiagen (238 mentions)
Qiaamp dna mini kit, by Qiagen (227 mentions)
Trizol reagent, by Takara Bio (225 mentions)
Primescript rt master mix, by Takara Bio (212 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (212 mentions)
Dnase 1, by Thermo Fisher Scientific (199 mentions)
Rna nano 6000 assay kit, by Agilent Technologies (199 mentions)
10 155 protocols using nanodrop 2000
1
Gene Expression Profiling of Granta-519 Cells
2
Microbial Community Profiling of Fermentation Stages
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The genomic DNA was extracted from a total of 21 samples taken from seven fermentation stages using Soil DNA Kit (Omega Bio-tek, Inc.) following the manufacturer’s protocol. The DNA quality and quantity were determined by NanoDrop 2000 (Thermo, USA). For prokaryotes, the V4 hypervariable region of the 16S rRNA genes was amplified using universal primer 515F and 909R [18 (link)]. For eukaryotes, the ITS2 region of fungal rRNA gene was amplified using universal primer ITS4 and ITS7 [19 (link)]. Primer 515F and ITS4 were added with barcodes. PCR conditions were described in detail previously [20 (link)]. The amplified PCR products were analyzed through a 1%(wt/vol) agarose gel and purified using a PCR purification kit (GE0101–50, TSINGKE). The concentrations of PCR purified products were assessed by NanoDrop 2000 (Thermo, USA). Subsequently, purified amplicons of all samples were equally pooled for constructing a PCR amplicon library, according to the protocols of the Illumina TruSeq. DNA sample preparation LT kit (San Diego, CA, USA), and then subjected to sequencing using the Illumina MiSeq platform at the Environmental Genomic Platform of the Chengdu Institute of Biology, CAS.
3
Total RNA and DNA Extraction
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Total RNA and DNA were isolated using Tri-Reagent extraction (Sigma–Aldrich, Munich, Germany) starting from 50–100 mg of tissues (depending on the initial specimen size). RNA quality and yield were verified by Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) using a RNA 6000 Nano Kit (Agilent Technologies, Ltd.) and NanoDrop 2000 (Thermo Scientific, NanoDrop products, Wilmington, USA). DNA purity was estimated using NanoDrop 2000 (Thermo Scientific, NanoDrop products, Wilmington, USA).
4
RNA Isolation from Cultured Cells
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For RNA isolation, the treated cells were first washed with 1 mL phosphate-buffered saline (PBS) (10010023, Gibco) per well and then harvested with 350 μL buffer RLT (74104, Qiagen, Germany) supplemented with 1% β-mercaptoethanol (β-ME). Afterwards, the RNA was isolated with an RNase Mini Kit (74104, Qiagen, Hilden, Germany) following the producer’s protocol including an on-column DNA digestion (RNase-Free DNase, Qiagen, Germany) and a DNase step for removal of genomic DNA. In order to control the success of the purification and to ensure a uniform cDNA synthesis, every sample was measured twice (Nanodrop 2000, Thermo Fisher Scientific, Waltham, MA, USA).
After isolation, according to the manufacturer’s instructions, the eluted RNA purity and quantity of each sample was verified photometrically by optical density (OD) readings of the A260/280 nm ratio (Nanodrop 2000, Thermo Fisher Scientific, USA). The spectrophotometric ratio of A260/280 varied >1.80 and A260/230 values yield a ratio >2.0. Thus, the isolated RNA could be regarded as free of protein and polyphenols/polysaccharides contaminants.
After isolation, according to the manufacturer’s instructions, the eluted RNA purity and quantity of each sample was verified photometrically by optical density (OD) readings of the A260/280 nm ratio (Nanodrop 2000, Thermo Fisher Scientific, USA). The spectrophotometric ratio of A260/280 varied >1.80 and A260/230 values yield a ratio >2.0. Thus, the isolated RNA could be regarded as free of protein and polyphenols/polysaccharides contaminants.
5
P. cineraria Genome Assembly and Transcriptome
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For the reference genome assembly, fresh leaves of P. cineraria were collected from one tree (~15–20 years old) growing in the desert of Sweihan area (Figure 1 A; 24°17′18.3″ N 55°43′36.2″ E), Al Ain, Abu Dhabi Emirates, UAE. Genomic DNA was extracted from the green leaves using a modified Cetyl trimethylammonium bromide (CTAB) method (detailed DNA isolation method is described in Supplementary Information A ). The quantity of the DNA was determined by NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA), and the quality was confirmed in a 1% (w/v) agarose gel.
To assist gene annotation, RNA was extracted from leaves, roots, or flowers using a modification of a previously published method [55 (link)] (the detailed RNA isolation method is described inSupplementary Information B ). The isolated RNA concentration was estimated by NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA), and quality was checked by 1.2% agarose gel. The extracted RNA was sent to the Yale Center for Genomic Analysis (YCGA) for library preparation and sequencing on Illumina Hiseq 2000 (Illumina, San Diego, CA, USA).
To assist gene annotation, RNA was extracted from leaves, roots, or flowers using a modification of a previously published method [55 (link)] (the detailed RNA isolation method is described in
6
Total RNA Extraction and cDNA Synthesis
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All DNA manipulations were performed by standard techniques (Sambrook et al., 1989 ). Total fungal and plant RNAs were extracted from the mycelia of each sample using TRIzol® RNA kit (Invitrogen Life Technologies, Carlsbad, CA, United States), according to the manufacturer’s instructions. RNA concentrations were determined using a NanoDrop 2000 (Thermo Scientific, Wilmington, DE, United States), and RNA integrity was verified using 1% agarose gel electrophoresis. cDNA was synthesized using 1 μg of total RNA and a reverse transcription system based on the use of an Oligo(dT)15 primer (Promega, Madison, WI, United States). cDNAs were quantified using a NanoDrop 2000 (Thermo Scientific, Wilmington, DE, United States).
7
Extracellular Vesicle Protein Quantification
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The concentration of the protein content was estimated by the absorbance reading at 280 nm (NanoDrop 2000, Thermo Fisher Scientific, Wilmington, DE, USA). The protein content of approximately 30 µg of EVs was reduced using dithiothreitol (10 mM final concentration, 3 h at 37 °C) and alkylated with iodoacetamide (25 mM final concentration, 30 min in the dark at room temperature). Extracted proteins were incubated with trypsin (Promega, Madison, WI, USA) 1:50 (w/w) for ~20 h at 37 °C and 45 min at 56 °C thermoblock (Eppendorf, Hamburg, Hamburg, Germany). The reaction was stopped by adding trifluoroacetic acid to a final concentration of 1% (v/v). The digested peptide mixture was desalted in reverse-phase homemade microcolumns with POROS R2 resin (Applied Biosystems, Waltham, MA, USA). Samples were dried completely in a vacuum centrifuge (Savant Speed Vac Plus SC210A; Thermo Fisher Scientific, Waltham, MA, USA) and resuspended with 1% formic acid. The concentration of the peptide mixture was estimated by the absorbance reading at 280 nm (NanoDrop 2000, Thermo Fisher Scientific, Wilmington, DE, USA). Samples were stored at −20 °C for MS analysis.
8
Goat Blood DNA and RNA Extraction
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Genomic DNA was extracted from the 569 goats blood samples according to the instructions of the Animal Blood DNA Extraction Kit (Tiangen, Beijing, China). The DNA was dissolved in enzyme-free sterile water (DNase/RNase-free ddH2O) (Tiangen, Beijing, China), the DNA sample concentration was measured with a NanoDrop 2000 (Thermo, Waltham, MA, USA), and a 1.2% agarose gel was used to detect DNA quality. The extracted DNA concentrations ranged from 300 to 500 ng/μL and were then diluted to 50 ng/μL for subsequent experiments.
Total RNA extraction was performed according to the instructions of the TRIzol kit (Tiangen, Beijing, China). RNA sample concentration and quality were detected with a NanoDrop 2000 (Thermo, Waltham, MA, USA) and 1.2% agarose gels. Then, the RNA was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, Kusatsu, Japan) and stored at −20 °C.
Total RNA extraction was performed according to the instructions of the TRIzol kit (Tiangen, Beijing, China). RNA sample concentration and quality were detected with a NanoDrop 2000 (Thermo, Waltham, MA, USA) and 1.2% agarose gels. Then, the RNA was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, Kusatsu, Japan) and stored at −20 °C.
9
RNA Extraction and Quantification Protocol
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Total RNAs were extracted using TRIzol reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol and then quantified using NanoDrop 2000 (Thermo Fisher Scientific, Inc.). The PrimeScript™ RT Reagent Kit (Takara Biotechnology Co., Ltd., Dalian, China) was utilized for RT-PCR. microRNAs were extracted using RNAiso reagent (Takara Biotechnology Co., Ltd.) and then quantified using NanoDrop 2000 (Thermo Fisher Scientific, Inc.). RT-qPCR was subsequently performed according to the SYBR® Premix Ex Taq II ™ kit instructions (Takara Biotechnology Co., Ltd.). The reaction conditions were as follows: 95°C for 30 sec; and 95°C for 5 sec, 55°C for 30 sec and 72°C for 30 sec for 40 cycles. Primers for the target genes are listed in Table II . For each sample, gene expression was analyzed using the 2−ΔΔCq method (25 (link)). The results were normalized against an internal control (U6 RNA for miRNAs or GAPDH for mRNA). All experiments were repeated at least three times.
10
RNA Extraction and cDNA Synthesis
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Total RNA was extracted from the control and treated roots with TRIzol® reagent and liquid nitrogen method with some modifications36 (link). The purity and concentration of RNA were determined using a Nanodrop 2000 (Thermo scientific). An equal concentration of RNA was used to synthesize cDNA by using the protocol of LUNA Universal one-step Reverse Transcriptase Polymerase Chain Reaction (RT-PCR Kit). Briefly, the master mixture consisted of dye (reaction mixture), enzyme mixture, water, and template. Then again quantification of cDNA was done on Nanodrop 2000 (Thermo scientific).
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