Genomic DNA extraction was conducted from about fifteen 1‐month‐old seedlings using a modified cetyltrimethylammonium bromide (CTAB) protocol that was originally published by Carroll et al. (1995 ). DNA quality was assessed using 0.7% (w/v) gel electrophoresis and NanoDrop 8000 UV‐Vis Spectrophotometers (Thermo Fisher Scientific, Wilmington, DE). DNA quantification was carried out using the Qubit® 3.0 Fluorometer (Life Technologies, Carlsbad, CA) with the dsDNA HS (High Sensitivity) Assay Kit. In order to constitute the two DNA bulks, equal amounts of DNA were bulked and the final concentration adjusted to about 70 ng/μL for both bulks.
Nanodrop 8000 uv vis spectrophotometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The NanoDrop 8000 UV-Vis Spectrophotometer is a laboratory instrument used for the quantification and characterization of nucleic acids and proteins. It utilizes microvolume sample analysis technology to measure the absorbance of small sample volumes directly, without the need for cuvettes or dilutions.
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Lab products found in correlation
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (7 mentions)
Applied biosystems 7900ht fast real time pcr system, by Thermo Fisher Scientific (6 mentions)
Maxwell 16 instrument, by Promega (4 mentions)
Fast advanced master mix, by Thermo Fisher Scientific (4 mentions)
Maxwell simplyrna kit, by Promega (4 mentions)
Oragene dna collection kit, by DNA Genotek (3 mentions)
Dneasy 96 plant mini kit, by Qiagen (2 mentions)
Applied biosystems taqman assays 20, by Thermo Fisher Scientific (2 mentions)
Dna isolation kit for cells and tissue, by Roche (2 mentions)
Massarray system, by Agena (2 mentions)
Maxwell simplyrna tissue kit, by Promega (2 mentions)
Lightcycler 480 sw version 1, by Roche (2 mentions)
Lightcycler 480, by Roche (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Sybr green, by Thermo Fisher Scientific (2 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Synergy ht multi mode microplate reader, by Agilent Technologies (1 mentions)
Epitect fast bisulfite conversion kit, by Qiagen (1 mentions)
Rotor gene q instrument, by Qiagen (1 mentions)
37 protocols using nanodrop 8000 uv vis spectrophotometer
1
Genomic DNA Extraction from Seedlings
2
Cultivated and Wild Grape Germplasm Collection
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A germplasm collection of 51 cultivated (V. v. spp. sativa) and 44 wild-type (V. v. spp. sylvestris) female grapevines was sorted at the FEM grape repository (ITA362), located in San Michele all'Adige, Italy (S1 Table ). The sativa accessions were chosen within a genetic core collection (G-110) that retains 100% of SSR and SNP loci diversity present in the source collection [30 (link)]. The wild individuals, mostly originating from the Italian Peninsula, were selected within the sylvestris accessions of the same repository previously clustered through a hierarchical STRUCTURE analysis [30 (link)]. Young leaf tissue of one field grown plant per accession was harvested and stored immediately in sterile tubes at -80°C for DNA extraction and successive analyses. Total genomic DNA was isolated from freeze-dried tissue after grinding with the MM 300 Mixer Mill system (Retsch., Germany) using the DNeasy 96 plant mini kit (QIAGEN, Germany). DNA concentration and purity were checked both by the Synergy HT Multi-Mode Microplate Reader (BioTek) and the NanoDrop 8000 UV-Vis Spectrophotometers (Thermo Scientific).
3
Quantitative DNA Methylation Assay
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DNA was extracted from 15mg tissue samples using the DNAeasy Blood & Tissue kit (Qiagen GmbH) according to the manufacturer’s instructions. DNA concentrations were estimated by measuring the absorbance at 260 nm (NanoDrop 8000 UV-Vis Spectrophotometer; Thermo Fisher Scientific, Victoria, Australia). A total of 25 ng of DNA in 20-µL distilled water was bisulphite converted and purified using the EpiTect Fast Bisulfite Conversion Kit (Qiagen). A duplex polymerase chain reaction (PCR) assay targeting methylated regions residing in the promoters of BCAT1 (promoter 1) and IKZF1 as previously described20 23 (link, link, link) was performed on a Rotor-Gene Q instrument (Qiagen) in a final PCR volume of 15 μL (50 cycles: 94°C, 60 seconds: 60°C, 60 seconds). A bisulphite-conversion specific ACTB PCR assay was performed separately as the control assay.20 23 (link, link, link) The level of methylation was expressed as mass of methylated BCAT1 or IKZF1 DNA as a percentage of the total amount of analysed DNA (ACTB).
4
Quantifying mRNA Expression Changes
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RNA was extracted using Maxwell simplyRNA Kits and a Maxwell 16 Instrument (Promega). RNA concentrations were measured using a NanoDrop 8000 UV-Vis Spectrophotometer (Thermo Fisher Scientific). All cDNA used in mRNA quantitative real-time PCR (qPCR) analyses was synthesized from extracted RNA by using the SuperScript VILO cDNA Synthesis Kit (Life Technologies) in accordance with the manufacturer’s protocol. The mRNA expression data were generated using Applied Biosystems TaqMan assays (20×) and Fast Advanced Master Mix (Life Technologies). Thermal cycling for qPCR was performed with an Applied Biosystems 7900HT Fast Real-Time PCR system (Life Technologies) in accordance with the TaqMan Fast protocol. Gene expression was normalized to the housekeeping gene 18 S ribosomal RNA (18 S), the expression of which did not vary in the different cell lines as a function of the glucose levels. Data are shown as the mRNA fold change (2−ΔΔCT) relative to the mRNA level of the corresponding transcript in the control samples as indicated. Each experiment was performed at least three times, and all samples were analyzed in triplicate.
5
RNA Extraction and Real-Time qPCR Analysis
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RNA was extracted using Maxwell simplyRNA Kits and a Maxwell 16 Instrument (Promega) as previously described15 (link),17 (link). Briefly, RNA concentrations were measured using a NanoDrop 8000 UV-Vis Spectrophotometer (Thermo Fisher Scientific). All cDNA used in mRNA quantitative real-time PCR (qPCR) analyses was synthesized from extracted RNA by using the SuperScript VILO cDNA Synthesis Kit (Life Technologies) in accordance with the manufacturer’s protocol. The mRNA expression data were generated using Applied Biosystems TaqMan assays (20×) and Fast Advanced Master Mix (Life Technologies, catalog number 4444556). Thermal cycling for qPCR was performed with an Applied Biosystems 7900 HT Fast Real-Time PCR system (Life Technologies) according to the TaqMan Fast protocol. Gene expression was normalized to the housekeeping gene 18S ribosomal RNA (18S), the expression of which did not vary as a function of the experimental conditions. Data are shown as the mRNA fold change (2−ΔΔCT) relative to the mRNA level of the corresponding transcript in the control samples as indicated. Each experiment was performed at least three times, and all samples were analyzed in triplicate.
6
Microarray-based Genome Profiling Protocol
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For each sample,
a predetermined amount of synthetic DNA was added to 1 μg of
extracted DNA as a spike-in control. Subsequently, the mixed DNA was
labeled with Cy-3 (or Cy-5) fluorescent dye (GE Healthcare, Vacaville,
CA, USA) using random primers and Klenow fragment of DNA polymerase
I. Labeled DNA was then purified using a QIAquick Purification kit
(Qiagen, Valencia, CA, USA), and the NanoDrop 8000 UV–vis Spectrophotometer
(Thermo Scientific; Waltham, MA) was used to measure the yield and
degree of labeling. Each sample was supplemented with a total of 42 μL
of buffer containing 1× HI-RPM hybridization buffer, 1×
aCGH blocking agent, 0.05 μg/μL of Cot-1 DNA, and 10%
formamide. The mixture was then vortexed thoroughly, spun down, and
incubated at 95 °C for 3 min, followed by incubation at 37 °C
for 30 min. The samples were subsequently hybridized with CyanoStrainChip
at 67 °C for 24 h with a rotation at 20 rpm in an Agilent hybridization
oven (Agilent Technologies, Inc., Santa Clara, CA, USA). For posthybridization
washing, an Agilent Wash Buffer Kit (Agilent, Santa Clara, CA) was
used for removing nonhybridized or partially hybridized labeled sample
DNA from the array’s surface to minimize signal noise.
a predetermined amount of synthetic DNA was added to 1 μg of
extracted DNA as a spike-in control. Subsequently, the mixed DNA was
labeled with Cy-3 (or Cy-5) fluorescent dye (GE Healthcare, Vacaville,
CA, USA) using random primers and Klenow fragment of DNA polymerase
I. Labeled DNA was then purified using a QIAquick Purification kit
(Qiagen, Valencia, CA, USA), and the NanoDrop 8000 UV–vis Spectrophotometer
(Thermo Scientific; Waltham, MA) was used to measure the yield and
degree of labeling. Each sample was supplemented with a total of 42 μL
of buffer containing 1× HI-RPM hybridization buffer, 1×
aCGH blocking agent, 0.05 μg/μL of Cot-1 DNA, and 10%
formamide. The mixture was then vortexed thoroughly, spun down, and
incubated at 95 °C for 3 min, followed by incubation at 37 °C
for 30 min. The samples were subsequently hybridized with CyanoStrainChip
at 67 °C for 24 h with a rotation at 20 rpm in an Agilent hybridization
oven (Agilent Technologies, Inc., Santa Clara, CA, USA). For posthybridization
washing, an Agilent Wash Buffer Kit (Agilent, Santa Clara, CA) was
used for removing nonhybridized or partially hybridized labeled sample
DNA from the array’s surface to minimize signal noise.
7
Genomic DNA Extraction from Blood
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Genomic DNA was extracted from whole blood samples using a modified salting out method described previously [35 (link), 36 (link)]. DNA samples were evaluated by spectrophotometry using the Thermo Scientific NanoDrop™ 8000 UV-Vis Spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE. USA) to determine DNA yield and 260/280 ratios [37 –39 ]. Samples with a reading below 1.7 for their 260/280 ratio were purified using an ethanol precipitation protocol to guarantee DNA sample purity [40 ].
8
Preparing KRAS mutation admixtures
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Cell line DNA was extracted from a frozen cell pellet containing ~5×106 cells using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). KRAS mutation admixtures were created by quantifying in triplicate the high molecular weight DNA using a NanoDrop 8000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Then, 1000 copies/µL DNA mutant standards were made from each cell line, which was diluted with known KRAS wild-type human genomic reference DNA (Roche Diagnostics, Mannheim, Germany) to create 10 distinct admixtures per mutation with mutant allele frequencies of 20%, 10%, 5%, 1% and 0.5% (table 2 ), assuming 50% allelic frequency with the heterozygous samples.
Two sets of admixtures were created for all concentrations: one with 100 copies/µL mutant allele and one with 50 copies/µL mutant allele, resulting in a total series of 50 cell line admixtures. In addition to the 50 cell lines admixtures, six wild-type control samples were prepared with total input DNA copy numbers equivalent to those in the cell line admixtures. Admixtures were prepared in Axygen Maxymum recovery 1.7 mL tubes (Corning, Wiesbaden, Germany). The six wild-type controls contained 400, 1900, 9900 and 19 900 copies/µL, respectively.
Two sets of admixtures were created for all concentrations: one with 100 copies/µL mutant allele and one with 50 copies/µL mutant allele, resulting in a total series of 50 cell line admixtures. In addition to the 50 cell lines admixtures, six wild-type control samples were prepared with total input DNA copy numbers equivalent to those in the cell line admixtures. Admixtures were prepared in Axygen Maxymum recovery 1.7 mL tubes (Corning, Wiesbaden, Germany). The six wild-type controls contained 400, 1900, 9900 and 19 900 copies/µL, respectively.
9
Quantifying UCHL1 Expression in Glioma Cells
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Total RNA was extracted from the cells using a phenol-free total RNA extraction kit (Norgen Biotek, ON, Canada). Quality and concentration of the extracted RNA was determined by spectrophotometric methods (NanoDrop 8000 UV-Vis Spectrophotometer, Thermo Scientific, Waltham, MA). The levels of expression of the UCHL1 and housekeeping gene (HPRT1) were measured in the glioma cell lines and in the UCHL1 KDs using droplet digital PCR (QX100 ddPCR System, Bio-Rad, Hercules, CA) and Taqman Gene Expression primer/probes sets (Human HPRT1 (FAM-labeled) and UCHL1 (VIC-labeled); Applied Biosystem, Foster City, CA). RT reactions were conducted on a Veriti PCR System (Applied Biosystems) using 100 ng of total RNA, and iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad) as per manufacturer’s guidelines (i.e. 5 min at 25°C, 30 min at 42°C, 5 min at 85°C followed by an end cycle at 4°C). Quantitative PCR reactions were prepared using 2 μL of the RT reactions (diluted 1/10), Taqman Gene Expression Assays, and ddPCR Supermix for probes (Bio-Rad) as per manufacturer’s guidelines. Differences in UCHL1 mRNA expression across samples were determined using QX100 Droplet Reader and QuantaSoft TM Software (Bio-Rad).
10
Genotyping of Vitis Germplasm Accessions
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A total of 90 Vitis accessions maintained at the INRAE repository of Vassal-Montpellier (Marseillan, France, www6.montpellier.inrae.fr/vassal_eng ) were used during this study (S1 Table ). This set included 13 Asian and 56 North American accessions representing 12 and 18 putative species, respectively. For Vitis vinifera we retained a set of eight cultivars (subsp. vinifera) amongst the most popular French ones and a set of eight wild accessions (subsp. sylvestris) mostly originated from French populations. Muscadinia rotundifolia was added as an outgroup with five accessions. Most of this germplasm has been studied and precisely identified [12 ] but seven accessions (THU2, YES, AES5, AES6, ARZ2, TRE, TRE2, S1 Table ) were more recently introduced to increase the number of species or the number of accessions for some species. Total genomic DNA was extracted from young leaf tissues using the DNeasy 96 plant mini kit (Qiagen Inc., Valencia, CA, USA). DNA concentration and purity was assessed using a NanoDrop 8000 UV-Vis spectrophotometer (Thermo Scientific).
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