Total RNA from S. microadriaticum was extracted by grinding the cell pellets with 200–300 μl of 0.5 mm glass beads (BioSpec Products, Bartlesville, OK) in liquid nitrogen-chilled mortar and pestle prior to using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The quality of extracted total RNA was assessed using a Bioanalyzer 2100 (Agilent, Santa Clara, CA). Strand-specific RNA-seq libraries were generated from oligo-dT selected total RNA using the NEBNext Ultra Directional RNA Prep Kit (New England Biolabs, Ipswich, MA) according to manufacturer's instructions. A total of 397 million paired-end read pairs (read length: 101 bp, insert size: 180 bp) were retrieved from 4 lanes on the HiSeq 2000 platform (Illumina, San Diego, CA).
Nebnext ultra directional rna prep kit
Manufactured by New England Biolabs
Sourced in United States
The NEBNext Ultra Directional RNA Prep Kit is a laboratory product designed for RNA library preparation. It is used to generate high-quality, strand-specific RNA sequencing libraries from a variety of input RNA sources.
Automatically generated - may contain errors
Lab products found in correlation
2100 bioanalyzer, by Agilent Technologies (4 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Rneasy plant mini kit, by Qiagen (2 mentions)
0.5 mm glass bead, by Biospec (2 mentions)
Hiseq 2000 machine, by Illumina (2 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Plant mini kit, by Qiagen (1 mentions)
Rna 6000 nano labchip, by Agilent Technologies (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Plant fungi total rna purification kit, by Norgen Biotek (1 mentions)
4 protocols using nebnext ultra directional rna prep kit
1
S. microadriaticum Total RNA Extraction
2
RNA Extraction from Algal Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RNA extractions, about 3 × 107 algal cells (200 ml of 1–2 × 105 cells/ml) were pelleted in 50 ml Falcon tubes at 500 g, decanted, washed, and re-pelleted at 1000 g. The pellets were drip-dried and subsequently snap-frozen with liquid nitrogen for subsequent processing. Total RNA was extracted from the cell pellets via grinding in liquid nitrogen. To aid grinding, 200–300 μl of 0.5 mm glass beads (BioSpec Products, Bartlesville, OK) were added into the mortar. Subsequently, the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) was used according to manufacturer’s instructions to extract RNA. The quality of RNA was assessed using a Bioanalyzer 2100 (Agilent, Santa Clara, CA) prior to sequencing library creation. Strand-specific RNA-Seq libraries were generated from oligodT-selected total RNA using the NEBNext Ultra Directional RNA Prep Kit (New England Biolabs, Ipswich, MA) according to manufacturer’s instructions. A total of 397 million paired-end read pairs (read length: 101 bp, library insert size: 180 bp) were sequenced and retrieved from the NCBI Short Read Archive (SRA) under BioProject accession PRJNA315758 [31 (link)].
3
Transcriptome Analysis of Wheat Domestication
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from glumes using the Plant Mini Kit including a digestion step with DNase I (Qiagen, Standford, CA, USA) for removal of DNA traces. High quality RNA was confirmed using Bioanalyzer 2100 with RNA 6000 Nano Labchips (Agilent, Santa Clara, CA, USA). RNA samples were pooled to three groups in accordance with their level of domestication, i.e., wild, landraces and cultivars. As the main objective of this study was to identify transcription differences along domestication “gradient”, pooling samples should give higher credence to representative genes of groups rather than genotypes. Each of the pools contained 1 μg RNA of the two accessions (Table 1 ). For each RNA pool, two independent biological replicates (i.e., six pools) were used to construct RNA-seq libraries, and a third replicate was reserved for QPCR validation. The cDNA libraries were constructed using NEBNext Ultra Directional RNA Prep Kit (New England Biolabs, MA), following the manufacturer’s instructions. After verifying the quality of the libraries indexed with six-nucleotide barcodes, sequencing was performed on the Illumina Hiseq2000 machine using multiplexing for generating 2 × 101 bp paired end reads. Sequencing was carried out at the Technion Genome Center (Haifa, Israel).
4
Transcriptomic Analysis of Plant Stress Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples of fresh shoot tissue were collected from treated and untreated plants at 24 HAT (Figure 1A ), immediately frozen in liquid nitrogen and stored at -80°C. RNA-seq analysis was conducted using three plants from each of the following treatments: control (C), pinoxaden application (X), high-temperature (H) and the combination of pinoxaden and high-temperature (HX; Figure 1A ). Total RNA was extracted using a Plant/Fungi Total RNA Purification Kit (Norgen Biotek Corp., Canada). Total RNA was treated with TURBODNase® (RNase-Free; Ambion, Warrington, United Kingdom) to eliminate DNA contamination. RNA was quantified using a NanoDrop (ND-1000) spectrophotometer (Thermo Scientific, Wilmington, DE, United States) and RNA integrity and quality were assessed with a 2100 Bioanalyzer (Agilent Technologies Inc., Germany). Additional data concerning sample quality and other parameters are presented in Supplementary Table S1 .
cDNA libraries were generated using a NEBNext Ultra Directional RNA Prep Kit (New England Biolabs, Ipswich, MA, United States), following the manufacturer’s instructions. After verifying their quality, libraries were indexed with six-nucleotide barcodes and sequencing was performed on the Illumina HiSeq2000 machine using multiplexing for generating 50 base-paired (bp) end reads. Sequencing was carried out at the Technion Genome Center (Haifa, Israel).
cDNA libraries were generated using a NEBNext Ultra Directional RNA Prep Kit (New England Biolabs, Ipswich, MA, United States), following the manufacturer’s instructions. After verifying their quality, libraries were indexed with six-nucleotide barcodes and sequencing was performed on the Illumina HiSeq2000 machine using multiplexing for generating 50 base-paired (bp) end reads. Sequencing was carried out at the Technion Genome Center (Haifa, Israel).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!