Mice aortas were pulverized using an automatic freeze-grinding device at −20 °C. Total RNA was extracted by Trizol reagent (CW0580S, Cwbio, Beijing, China). NanoDrop 2000 microvolume spectrophotometers (Thermo Scientific, Waltham, MA, USA) were used to measure RNA concentration and purity. The following manufacturer’s instructions were strictly followed for the reverse transcription and real-time PCR procedures. With the guidance of HiScript III RT SuperMix (R323-01, Vazyme, Nanjing, China), RNA (1 μg) isolated from aorta samples was converted into cDNA. Real-time PCR was conducted using Taq Pro Universal SYBR qPCR Master Mix (Q712-02, Vazyme). The relative gene expression adopted the calculation method of 2−ΔΔCt. Table S1 details the primer sequences (Actb, Tnf-α, Il-6, Il-1β, and Mcp-1), which were synthesized by Beijing Genomics Institution (Beijing, China).
Nanodrop 2000 microvolume spectrophotometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NanoDrop 2000 microvolume spectrophotometer is a compact and efficient instrument for quantifying and analyzing small-volume samples. It utilizes a patented sample retention system to measure the absorbance of samples as low as 0.5 μL, allowing for precise analysis of precious or limited-quantity samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Trizol reagent, by CWBIO (2 mentions)
Taq pro universal sybr qpcr master mix, by Vazyme (2 mentions)
Hiscript 3 rt supermix, by Vazyme (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Bca assay, by Merck Group (1 mentions)
Primescript 2 first strand cdna synthesis kit, by Takara Bio (1 mentions)
Rnaprep pure polysaccharide polyphenol plant total rna extraction kit, by Tiangen Biotech (1 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (1 mentions)
Hiseq 2500 sequencing system, by Illumina (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Fruit mate for rna purification, by Takara Bio (1 mentions)
Avance 500, by Bruker (1 mentions)
Sonopuls hd 2070, by Bandelin (1 mentions)
Glomax multi microplate multimode reader, by Promega (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
24 protocols using nanodrop 2000 microvolume spectrophotometer
1
Aorta Gene Expression Analysis
2
Aorta Gene Expression Analysis
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Mice aortas were pulverized using an automatic freeze-grinding device at −20 °C. Total RNA was extracted by Trizol reagent (CW0580S, Cwbio, Beijing, China). NanoDrop 2000 microvolume spectrophotometers (Thermo Scientific, Waltham, MA, USA) were used to measure RNA concentration and purity. The following manufacturer’s instructions were strictly followed for the reverse transcription and real-time PCR procedures. With the guidance of HiScript III RT SuperMix (R323-01, Vazyme, Nanjing, China), RNA (1 μg) isolated from aorta samples was converted into cDNA. Real-time PCR was conducted using Taq Pro Universal SYBR qPCR Master Mix (Q712-02, Vazyme). The relative gene expression adopted the calculation method of 2−ΔΔCt. Table S1 details the primer sequences (Actb, Tnf-α, Il-6, Il-1β, and Mcp-1), which were synthesized by Beijing Genomics Institution (Beijing, China).
3
Seed Starch Synthesis in Hulless Barley Cultivars
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Two elite hulless barley cultivars Zangqing 2000 (Q) and 08-1127 (C2) were conserved by the Tibet Academy of Agriculture and Animal Husbandry Sciences and used for gene analysis associated with seed starch synthesis. Zhangqing2000 (Q) has a higher amylose content (68.5%) and β-glucan content (6.88%) as compared to 08-1127 (C2), which has almost 100% amylose content and 11.23% β-glucan (data collected from 2012 to 2013 in Chengdu). The hulless barley plants were cultivated in test plots and grown under normal conditions in three experimental fields in Chengdu, Sichuan Province of China.
After hulless barley booting, grains of Zangqing 2000 (Q) and 08-1127 (C2) plants at 6, 8, 10, 12, 14, 16, 18, and 20 days after pollination (DAP) for RNA extraction were harvested as described in previous studies (Chen et al. 2014 (link)). Each sample consisted of grains from at least five individuals and pooled for each biological replicate. Total RNA samples were prepared using Trizol Reagent (Invitrogene, Nottingham, UK), in three replicates, according to the manufacturer’s instructions. The concentration and quality of RNA samples were determined using a NanoDrop 2000 micro-volume spectrophotometer (Thermo Scientific, Waltham, MA, USA). Equal amounts of RNA from each sample of the identical accessions were pooled to construct two complementary DNA (cDNA) libraries.
After hulless barley booting, grains of Zangqing 2000 (Q) and 08-1127 (C2) plants at 6, 8, 10, 12, 14, 16, 18, and 20 days after pollination (DAP) for RNA extraction were harvested as described in previous studies (Chen et al. 2014 (link)). Each sample consisted of grains from at least five individuals and pooled for each biological replicate. Total RNA samples were prepared using Trizol Reagent (Invitrogene, Nottingham, UK), in three replicates, according to the manufacturer’s instructions. The concentration and quality of RNA samples were determined using a NanoDrop 2000 micro-volume spectrophotometer (Thermo Scientific, Waltham, MA, USA). Equal amounts of RNA from each sample of the identical accessions were pooled to construct two complementary DNA (cDNA) libraries.
4
Extracting Total RNA from Prefrontal Tissues
5
Proteome Extraction and Analysis from Mini-Pig Cartilage
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Protein was extracted from mini-pig knee articular cartilage tissue samples by lysis using SDT buffer consisting of 4% (w/v) sodium dodecyl sulfate (SDS), 100 mM Tris-HCl (pH 7.6), and 0.1 M dithiothreitol (DTT) (Sigma-Aldrich, St. Louis MO, USA). The protein concentration was detected using the bicinchoninic acid (BCA) assay (Sigma-Aldrich, St. Louis MO, USA). The filter-aided proteome preparation (FASP) method was used for each sample containing an appropriate amount of protein, and the samples underwent trypsin digestion. The OD280 value (peptide content) was determined using the NanoDrop™ 2000 microvolume spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The quality of the total protein extracted from mini-pig knee cartilage tissues was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Sigma-Aldrich, St. Louis MO, USA) and tandem mass tag (TMT)-labeling liquid chromatography with tandem mass spectrometry (LC-MS-MS) (Thermo Fisher Scientific, USA). Each sample of 100 μg of the peptide was tagged using the Tandem Mass Tag™ 6-plex kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions.
6
Cloning Vacuolar Chloride Channel in Upland Cotton
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Total RNA was isolated from TM-1 seedlings using the RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (Tiangen, Beijing, China). The RNA concentration was determined using the NanoDrop 2000 microvolume spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States), whereas RNA integrity was assessed by 1% agarose gel electrophoresis. First-strand cDNA was synthesized from 1 μg RNA using the PrimeScriptTM II first Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). To clone the vacuolar chloride channel gene in upland cotton, the BlastP and tBlastN programs were performed against the G. hirsutum genome database1 using the A. thaliana CLCg amino acid sequence as the query. The coding sequences of gene IDs GH_A06G0574 and GH_D06G0541 as the best match were retrieved and gene-specific primers were designed. Full-length cDNAs were amplified using the GhCLCg-1F/R primer pair (Supplementary Table 1 ) and the KOD-Plus-Neo DNA polymerase (TaKaRa, Dalian, China). The PCR products were purified using the FastPure Gel DNA Extraction Mini Kit (Vazemy Biotech Co., Ltd.) and then cloned using the pEASY®-Blunt Cloning Kit for the subsequent transformation of Trans1-T1 Escherichia coli competent cells (TransGen Biotech). Positive clones cultured at 37°C were analyzed by sequencing to verify they were correctly transformed.
7
Comprehensive Pig Genome Resequencing
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We totally resequenced 55 samples which were all from livestock breeding station of Xiantan city (Hunan province, China), including 20 Shaziling pigs (S), 10 Berkshire pigs (BKX), 20 Dabasha pigs (DBS) and 5 Basha pigs (BS) in this study. We used TIANamp Genomic DNA Kit (DP304, TIANGEN) to extract genomic DNA from pig ear tissue. Quality, integrity, and concentration of extracted DNA were detected by 1.5% agarose gel electrophoresis and Thermo Scientific Nanodrop 2000 microvolume spectrophotometer. All qualified genomic DNA was resequenced using Illumina HiseqTM 2500 sequencing system. The Illumina DNA libraries (paired-end, 2*150 bp) were constructed for 55 pig samples using at least 1 μg genomic DNA with standard protocols and 2732.22 Gb raw data were generated in 10 × or 30 × sequencing depth. Further, we downloaded 11 Yorkshire pigs and 1 Warthog whole genome data from European Nucleotide Archive (ENA) database and totally we got 517.23 Gb raw data.
8
Nucleic Acid and Protein Extraction Protocol
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Genomic DNA was extracted from each cell line using DNeasy Blood and Tissue Kit
(Qiagen, Cat.No. 69504) according to the manufacturer’s instructions. DNA
concentrations were verified using the NanoDrop 2000 micro-volume spectrophotometer
(Thermo Scientific). Satisfactory DNA purity was regarded as greater than or equal to
a 260/280 ratio of 1.8, ensuring minimal protein contamination of the sample. The
quality of the DNA samples was further assayed using agarose gel electrophoresis.
After electrophoresis, the gel was carefully removed and the DNA bands were
visualised using the Gel Documentation System.
Total RNA was extracted from the cell lines in duplicate using the RNeasy MinElute
Cleanup Kit (Qiagen, Cat. no. 74204) and miRNeasy Mini Kit (Qiagen, Cat. no. 217004).
The concentration of the RNA was verified using the NanoDrop 2000 spectrophotometer.
Satisfactory RNA purity was regarded as a 260/230 ratio of approximately 2.0.
Protein lysates were prepared when the cell lines were approximately 80% confluent,
as described in detail elsewhere [20 (link)]. The protein concentration of the lysates was determined via the
bicinchoninic acid (BCA) assay (Sigma-Aldrich, cat. no. C2284-25ML, cat.no.
B9643-1L).
(Qiagen, Cat.No. 69504) according to the manufacturer’s instructions. DNA
concentrations were verified using the NanoDrop 2000 micro-volume spectrophotometer
(Thermo Scientific). Satisfactory DNA purity was regarded as greater than or equal to
a 260/280 ratio of 1.8, ensuring minimal protein contamination of the sample. The
quality of the DNA samples was further assayed using agarose gel electrophoresis.
After electrophoresis, the gel was carefully removed and the DNA bands were
visualised using the Gel Documentation System.
Total RNA was extracted from the cell lines in duplicate using the RNeasy MinElute
Cleanup Kit (Qiagen, Cat. no. 74204) and miRNeasy Mini Kit (Qiagen, Cat. no. 217004).
The concentration of the RNA was verified using the NanoDrop 2000 spectrophotometer.
Satisfactory RNA purity was regarded as a 260/230 ratio of approximately 2.0.
Protein lysates were prepared when the cell lines were approximately 80% confluent,
as described in detail elsewhere [20 (link)]. The protein concentration of the lysates was determined via the
bicinchoninic acid (BCA) assay (Sigma-Aldrich, cat. no. C2284-25ML, cat.no.
B9643-1L).
9
Transcriptome Analysis of Hulless Barley
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Two local varieties of Tibetan hulless barley, XQ754 and Nimubai (used and known as tribute barley), were conserved by the Tibet Academy of Agricultural and Animal Husbandry Sciences. Nimubai has a higher amylose content (33.9%) and β-glucan content (7.5%) as compared to XQ754, which had 27.2% amylose and 6.0% β-glucan (data collected from 2009–2010 in Chengdu). The hulless barley plants were cultivated in October, 2010 and grown under normal conditions in the three fields in Chengdu, Sichuan Province of China.
Grains of Nimubai and XQ754 plants were sampled at 5, 10, 15, 20, and 25 days after pollination (dap) for RNA extraction. Each sample consisted of grains from nine individuals. Total RNA was extracted from the grains using Trizol Reagent (Takara) and Fruit-mate for RNA purification (Takara), according to the manufacturer's instructions. The concentration and quality of RNA samples were determined using a Nano Drop 2000 micro-volume spectrophotometer (Thermo Scientific, Waltham, MA, USA). Equal amounts of RNA from each sample of the identical accessions were pooled to construct two cDNA libraries [26] (link), [27] (link).
Grains of Nimubai and XQ754 plants were sampled at 5, 10, 15, 20, and 25 days after pollination (dap) for RNA extraction. Each sample consisted of grains from nine individuals. Total RNA was extracted from the grains using Trizol Reagent (Takara) and Fruit-mate for RNA purification (Takara), according to the manufacturer's instructions. The concentration and quality of RNA samples were determined using a Nano Drop 2000 micro-volume spectrophotometer (Thermo Scientific, Waltham, MA, USA). Equal amounts of RNA from each sample of the identical accessions were pooled to construct two cDNA libraries [26] (link), [27] (link).
10
NMR Spectroscopy and Luminescence Assay
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1H–NMR spectra were recorded in CDCl3 (isotopic enrichment 99.95%) solutions at 300 K using a Bruker AVANCE 500 instrument (500.13 MHz for 1H, 125.76 MHz for 13C) using 5 mm inverse detection broadband probes and deuterium lock. Chemical shifts (δ) are given as parts per million relative to the residual solvent peak (7.26 ppm for 1H and 77.0, central line, for 13C) and coupling constants (J) are in Hertz. For two-dimensional experiments (COSY-45, HSQC, and HMBC), standard Bruker microprograms using gradient selection (gs) were applied (for more information, see electronic Supplemental Data ).
The quantification of the cDNA plasmid was done with a NanoDrop™ 2000 Microvolume Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and the sonicator was a Sonopuls HD2070 (Bandelin, Berlin, Germany) at ~50% power (~35 W). Luminescence signals (λmax = 559 nm) were recorded by a GloMax®-Multi+ Microplate Multimode Reader (Promega, Fitchburg, WI, USA). The GloMax reagent lines and injector were cleaned before and after use with 70% ethanol, then abundantly rinsed with ddH2O water.
The quantification of the cDNA plasmid was done with a NanoDrop™ 2000 Microvolume Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and the sonicator was a Sonopuls HD2070 (Bandelin, Berlin, Germany) at ~50% power (~35 W). Luminescence signals (λmax = 559 nm) were recorded by a GloMax®-Multi+ Microplate Multimode Reader (Promega, Fitchburg, WI, USA). The GloMax reagent lines and injector were cleaned before and after use with 70% ethanol, then abundantly rinsed with ddH2O water.
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