Total RNA was extracted from 100 mg of ripe fruits using the Nucleo-spin RNA Plant mini kit (Macherey-Nagel). Library preparation and Nanopore sequencing were performed at the Ecole normale superieure genomic core facility (Paris, France). After checking RNA quality by Fragment Analyzer, 10 ng of total RNA was amplified and converted into cDNA using SMART-Seq v4 Ultra Low Input RNA kit (Clontech). About 17 fmol of cDNA was used for library preparation using the PCR Barcoding kit (SQK-PBK004 kit, ONT) and cleaned up with 0.6× Agencourt Ampure XP beads. About 2 fmol of the purified product was amplified during 18 cycles, with a 17-min elongation step, to introduce barcodes. Samples were multiplexed in equimolar quantities to obtain 20 fmol of cDNA, and the rapid adapter ligation step was performed. Multiplexed library was loaded on an R9.4.1 flowcell (ONT) according to the manufacturer’s instructions. A standard 72-h sequencing was performed on a MinION MkIB instrument. MinKNOW software (version 19.12.5) was used for sequence calling. Long-reads were mapped on the tomato reference genome (SL2.5) using minimap267 (link) V2.11-r797 and visualized with IGV.
Nucleospin rna plant mini kit
Manufactured by Macherey-Nagel
Sourced in Germany
The NucleoSpin® RNA Plant mini kit is a laboratory tool designed for the isolation and purification of RNA from plant samples. It provides a reliable and efficient method to extract high-quality RNA for further analysis and applications.
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Lab products found in correlation
Smart seq v4 ultra low input rna kit, by Takara Bio (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Protoscript 2, by New England Biolabs (1 mentions)
Rnalater, by Merck Group (1 mentions)
Superscript 3 supermix, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Cfx96 system, by Bio-Rad (1 mentions)
Lightcycler 480 instrument, by Roche (1 mentions)
Rnase free dnase, by Qiagen (1 mentions)
Sensimix sybr green, by Meridian Bioscience (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
8 protocols using nucleospin rna plant mini kit
1
Nanopore Sequencing of Tomato Fruit Transcriptome
2
Transcriptomics Analysis of Immature Embryos
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Immature embryos of the same greenhouse grown plants as the biomass and metabolomics analyses were used to collect RNA for the transcriptomics analysis. For each of the three genotypes of WD‐1, WD‐2, and WT, RNA was extracted at four timepoints of 24, 27, 30, and 33 DAP. For each replicate, one immature embryo was flash‐frozen with liquid nitrogen and ground with pestle and mortar into fine powder from which RNA was isolated and cleaned using the Macherey‐Nagel NucleoSpin RNA Plant Mini kit (MACHEREY‐NAGEL Inc.). RNA samples were submitted for sequencing via the Illumina NovaSeq 6000 platform with 150 bp paired‐end reads at the Genomics Shared Resource of the Ohio State University Comprehensive Cancer Center.
3
Poplar Hydroponic Osmotic Stress Protocol
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Cuttings of Populus deltoides (Bartr.) Marsh x P. nigra L., clone Dorskamp, were grown in hydroponic conditions according to [61 (link)] with standard medium (MS). Osmotic stress was imposed to poplars by supplementing the medium with polyethylene glycol (PEG) 6000 at 50 g/L for 10 min. Roots, stems, petioles and leaf blades in control and stressed conditions were then harvested and frozen in liquid nitrogen. Total RNAs were extracted using the NucleoSpin® RNA Plant mini kit (Macherey-Nagel, Düren, Germany) according to manufacturer’s protocol, and reverse transcription was done with ProtoScript II (New England Biolabs) using one µg of RNAs. Specific HK1b primers were used at 0.2 µM for amplification of one µL of cDNAs by PCR (32 cycles). Clathrin cDNA expression was used as the amplification control (27 cycles) and PCRs were done in triplicate, using cDNAs from two biological experiments.
4
Transcriptomics of Specialized Ant-Plant Domatia
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Samples of three S. imberbis individuals were collected on Vanua Levu island in Fiji, along the Cross Island road before the bifurcation to Nabouwalu and Labasa in July 2018. Samples were immediately placed in RNAlater (Sigma‐Aldrich) until their extraction <72 h later. Plants were identified by the senior author (G.C.), following the taxonomy of Chomicki and Renner (2016b (link)) and documented by the herbarium voucher G. Chomicki, J. Aroles, A. Naikatini 50 (M). Three tissue samples per plant were collected: (1) the warty surface of the basal wall of the domatium (1 mm thickness); (2) the smooth surface in the apex cavities of the domatium (1 mm thickness); and (3) the tuber tissue under the wall surface. Tuber tissue (i.e., domatium tissue without any differentiation, shown in gray in Figure 1b ) was used as a control. All individuals sampled were healthy, mature plants of a similar size (domatium diameter c. 15 cm), each inhabited by P. nagasau ants (coming from distinct ant colonies). RNA extraction was performed using a NucleoSpin RNA Plant Mini Kit (Macherey‐Nagel, Düren, Germany) following the manufacturer's protocol.
5
RNA Extraction and qRT-PCR Analysis
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For gene expression analysis, RNA was extracted with the RNeasy Plant Mini Kit (Qiagen) or the NucleoSpin RNA Plant Mini Kit (Macherey & Nagel) according to the manufacturers' instructions and including an on‐column treatment with RNase‐free DNase (Qiagen). Samples of 1 μg RNA were reverse transcribed with SuperscriptIII SuperMix (Invitrogen) according to the manufacturer's instructions. Real‐time quantitative PCR (qRT‐PCR) analysis was performed in a final volume of 10 μL using the Power SYBR Green PCR Master Mix (Applied Biosystems) and following the protocol of the supplier. Reactions were run in a CFX96 system (Bio‐Rad) or a LightCycler 480 instrument (Roche). Expression data for Arabidopsis genes were extracted from the eFP Browser (Winter et al., 2007 ).
6
Populus Deltoides × Nigra Transcripts
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Cuttings of Populus deltoides (Bartr.) Marsh × P. nigra L., clone Dorskamp, grown in hydroponic conditions were used. After the collection of cuttings, the roots, stems, petioles, and leaf blades were then harvested and frozen in liquid nitrogen. Total RNAs were extracted using the NucleoSpin® RNA Plant mini kit (Macherey-Nagel) according to the manufacturer’s protocol, and reverse transcription was done with the M-MuLV Reverse Transcriptase RNase H- (Finnzyme) using one µg of RNAs. Specific primers for each HK (Table S2 ) were used at 0.2 µM for amplification of 1 µg of cDNAs by PCR (35 cycles). For HPt amplification, 30 or 35 PCR cycles were used and Clathrin cDNA expression was used as an amplification control with 30 cycles.
7
Quantitative Real-Time PCR for Transcripts
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RNA was extracted from seedling leaf material (Zadoks code 15, which corresponds to five visible leaves), using a Nucleospin RNA plant mini-kit (Macherey-Nagel, Germany). Leaf material (70–100mg) was ground to a powder in liquid N2 and resuspended in 400 μl of RA1 extraction buffer, containing 1% (w/v) PVP-40. Following clarification by centrifugation, the supernatant was processed according to the manufacturer’s instructions. Poly(dT) cDNA was prepared from 1mg of total RNA with SuperScript III reverse transcriptase (Invitrogen), using primers as specified in Supplementary Table S1 at JXB online. Quantitative PCR was performed using SYBR-green Sensimix (Bioline), in 384-well optical reaction plates with a Roche LightCycler 480 apparatus (Roche/Applied Biosystems). PCR conditions and quantification were as specified by the manufacturer (Roche). The relative number of copies obtained for each transcript was normalized against HvELF1 and HvTubulin (Nicot et al., 2005 (link); Jarošová and Kundu, 2010 (link)) or AtCTL1 and AtCTL3 (De Rybel et al., 2010 (link)) transcript values for each sample as an internal reference.
8
Poplar RNA Extraction and Analysis
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This study was performed using the poplar clone 'Dorskamp'. Roots, stems, petioles and leaf blades of one month-old hydroponically grown rooted cuttings [40] were harvested and frozen. RNA extractions were carried out using the NucleoSpin RNA Plant mini kit (Macherey-Nagel). One μg of total RNA was reverse transcribed using M-MuLV Reverse Transcriptase RNase H-(Finnzyme), according to the manufacturer's procedure, and used as a template for PCR amplifications. Thirty or forty PCR cycles were performed to detect dkRRA transcripts, and clathrin was used as an expression control gene. The amplified fragments were separated by 1.2% agarose gel electrophoresis, stained with ethidium bromide, and analyzed under UV light. All PCRs were performed in triplicate at least, and three independent biological replicates were performed.
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