For each of six perennial species a single seed of one accession was scarified and planted in a 1:1 soil and sand mix and grown in the greenhouse. Leaves, pods, and flowers were harvested from single plants and roots were harvested from 5 to 10 seedlings grown on a wet paper towel in a petri-dish for 3 to 7 days. Total RNA was extracted from leaf, pod, flower and root tissue from each of the six perennial species using the Qiagen mini-RNA prep (Qiagen, Valencia, CA). Poly-A+ RNA was isolated from the total RNA using a Dynabead mRNA purification kit (Invitrogen, Carlsbad, CA) and cDNA libraries were constructed following the protocol “Preparing samples for sequencing of mRNA” (Illumina Inc., San Diego, CA).
Dynabead mrna purification kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Dynabeads mRNA Purification Kit is a laboratory equipment used to isolate and purify messenger RNA (mRNA) from a variety of sample types, including cells, tissues, and bodily fluids. The kit utilizes magnetic beads coated with oligo(dT) to selectively capture and isolate poly(A)-tailed mRNA molecules, allowing for efficient separation from other RNA species and contaminants.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Hiseq 2500, by Illumina (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Qiazol lysis reagent, by Qiagen (2 mentions)
Superscript 4, by Thermo Fisher Scientific (2 mentions)
Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Superscript reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Nextera transposase, by Illumina (1 mentions)
Norgen animal tissue rna purification kit, by Norgen Biotek (1 mentions)
M6a rna methylation quantification kit, by Abcam (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nebnext multiplex oligos, by New England Biolabs (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Rneasy midi kit, by Qiagen (1 mentions)
Rna 6000 nano chip, by Agilent Technologies (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Superscript 3 rt kit, by Thermo Fisher Scientific (1 mentions)
Agencourt ampure xp bead, by Beckman Coulter (1 mentions)
11 protocols using dynabead mrna purification kit
1
Transcriptome Analysis of Perennial Species
2
RNA-seq Transcriptome Profiling Protocol
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RNA-seq was performed as previously outlined [50 ] using Nextera transposases (Illumina). Briefly, cells were lysed with Buffer RLT (Qiagen) containing 10 % beta-mercaptoethanol. The Norgen Animal Tissue RNA Purification Kit (Norgen Biotek) was used to isolate RNA from cells. The Dynabead mRNA Purification Kit (Invitrogen) beads were used to isolate mRNA transcripts and cDNA synthesis was performed using Superscript reverse transcriptase (Invitrogen). Nextera transposases (Illumina) were used to fragment cDNA prior to next-generation sequencing. All experimental treatments at all time points were performed in quadruplicate.
3
Quantitative m^6A RNA Methylation Assay
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Total RNA from pulmonary cells or lung tissues was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The mRNA was purified from total RNA using the Dynabead mRNA Purification Kit (Invitrogen, Carlsbad, CA, USA). Two hundred nanograms of total RNA or mRNA were measured with a m6A RNA Methylation Quantification Kit (Abcam, Cambridge, MA, USA) according to the manufacturer's instructions. In brief, the test wells were coated with 200 ng total RNA or mRNA. Dilutions of the capture antibody solution and detection antibody solution were then added to each test well, and the optical absorbance of the samples was measured at 450 nm.
4
Transcriptomic Profiling of Fetal Liver
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Total RNA was extracted and purified from E14.5 fetal liver using the Qiagen RNeasy Mini Kit (Qiagen, 74104) according to manufacturer’s instructions. Polyadenylated messenger RNA (mRNA) was selectively enriched using Invitrogen Dynabead mRNA Purification Kit (Invitrogen, 61006). A sequencing library was constructed with NEBNext Ultr II RNA Library Prep Kit (NEB, E7770S) and amplified with NEBNext Multiplex Oligos (NEB, E7335S). Paired-end reads were obtained with Illumina HiSeq2500. Paired-end reads were aligned to the mouse genome (Gencode vM14) with Tophat and transcripts were assembled with Cufflinks64 (link).
5
Leaf RNA-seq and Gas Exchange Protocols
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Sampling of leaf material forRNA‐seq was linked to the gas exchange measurements via sampling paired leaves at the same developmental stage and canopy position (selecting the third and fourth fully expanded leaves from the growth tip in the upper canopy fully exposed to light) sampled at the same time of day (1000–1200). Leaf discs (three 6.3 mm diameter Sigma‐Aldrich cork‐borer) were sampled from an intact leaf, and immediately immersed into liquid nitrogen before storage at −80°C. Total RNA was extracted using a modified cetyltrimethylammonium bromide method specified for plant species (White et al., 2008 (link)).
Library preparation and sequencing were performed by the Next‐generation sequencing facility at Western Sydney University. In brief, total RNA samples were first treated with DNase I following the manufacturer's protocol (Qiagen), before poly(A) mRNA selection library preparation was carried out using oligo d(T) magnetic beads (Dynabead mRNA purification kit; Invitrogen). The mRNA was then fragmented at 94°C using divalent cations, and each fragment of mRNA was synthesized to cDNA. Sequencing adapters were then ligated to each fragment, amplified by PCR, and purification of amplified cDNA molecules. Sequencing took place on an Illumina HiSeq. 2000 (Illumina Inc.), producing 100‐bp paired‐end reads.
Library preparation and sequencing were performed by the Next‐generation sequencing facility at Western Sydney University. In brief, total RNA samples were first treated with DNase I following the manufacturer's protocol (Qiagen), before poly(A) mRNA selection library preparation was carried out using oligo d(T) magnetic beads (Dynabead mRNA purification kit; Invitrogen). The mRNA was then fragmented at 94°C using divalent cations, and each fragment of mRNA was synthesized to cDNA. Sequencing adapters were then ligated to each fragment, amplified by PCR, and purification of amplified cDNA molecules. Sequencing took place on an Illumina HiSeq. 2000 (Illumina Inc.), producing 100‐bp paired‐end reads.
6
Transcriptome Profiling of Khat and Ephedra
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Young khat leaves, approximately 1–3 cm in length, were harvested during daylight hours, and total RNA was isolated using an RNeasy Midi kit (Qiagen). For E. sinica, freshly emerging, light green “young” stems up to 5 cm in length were harvested for total RNA extraction using essentially the same procedure as for the khat tissue. Poly(A)+ RNA purification, cDNA library preparation, emulsion-based PCR (emPCR) and sequencing were performed at the McGill University and Génome Québec Innovation Center (Montréal, Canada). RNA quality was assessed using an RNA 6000 Nano chip on a BioAnalyzer 2100 (Agilent Technologies) to ensure an RNA Integrity Number (RIN) of > 7.5. Poly(A)+ RNA was prepared using a Dynabead mRNA Purification kit (Invitrogen). cDNA library preparation and Illumina GA sequencing were performed as described [18 (link)].
7
Bulk RNA-Seq of Synchronized C. elegans
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50,000 synchronized adult animals (~68 h at 20 °C after L1 arrest) were collected, in biological triplicates, by washing worms off plates using water and allowing the animals to settle on ice to generate a compact pellet. Worm pellets were resuspended in 1 ml TRIzol reagent (Thermo Fisher 15596018) and freeze-thawed on dry ice followed by vortexing. RNA isolation was performed by chloroform extraction and isopropanol precipitation. RNA samples were normalized to 7.5 μg per 10 μL, followed by polyA selection using the Dynabead mRNA Purification Kit (Invitrogen 61006). 100 ng of polyA selected mRNA samples were treated using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB E7760S) according to the manual, using NEBNext multiplex oligos for Illumina (NEB E7335S). mRNA libraries were sent to USC sequencing core for quality control (Agilent BioAnalyzer Chip) and sequenced on the Illumina NextSeq2000 100-cycle (SE 75-bp reads) platform. Primer sequences are available in Supplementary Data 3 . Differentially expressed gene lists can be found in Supplementary Data 6 . Sequencing library statistics summary can be found in Supplementary Data 5 .
8
RNAseq Library Enrichment for Low-Abundance Transcripts
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RNAseq libraries enriched for low-abundance transcripts were prepared for AML cell lines, lymphomas, and FACS-sorted PreB and ProB cells as follows. RNA was extracted using Trizol reagent (Thermo Fisher Scientific), DNase-treated, then polyadenylated transcripts were isolated using the Dynabead mRNA purification kit (Thermo Fisher Scientific). Purified mRNA was fragmented by heating to 98°C for 30 min in RNA storage buffer (Ambion), then converted to cDNA with random primers using the Superscript III RT kit (Invitrogen). Second-strand synthesis was performed in the presence of dUTP, then Illumina adapters were ligated onto the dsDNA fragments. To preserve strand identity, the uracil-containing cDNA strand was digested using USER enzyme (NEB), then cDNA was amplified using adapter-specific PCR primers and purified using Agencourt AMPure XP beads (Beckman Coulter). The samples were subsequently enriched for low-abundance transcripts by Duplex-Specific Nuclease (DSN) treatment (Illumina) followed by low-cycle PCR, then gel-purified and cleaned up using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). Libraries were sequenced by paired-end 76 bp on an Illumina HiSeq2500.
9
RNA-seq Library Preparation and Analysis
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Cells were lysed with Buffer RL (Norgen) containing 10% beta-mercaptoethanol. Cell lysate was collected after addition of 300 μl 100% molecular biology grade ethanol. Total RNA was extracted using the Animal Tissue RNA Purification Kit (Norgen). RNA-seq libraries were prepared from 250 ng total RNA via polyA-selection (Dynabead mRNA Purification Kit, ThermoFisher Scientific) followed by transposase-mediated non-stranded library construction [25 (link)]. Each experimental treatment was performed in triplicate. Libraries were pooled and sequenced on an Illumina HiSeq 2000 sequencer using paired-end 50 bp reads with a 6 bp index read (Illumina, Inc., San Diego, CA). Pooled sequencing resulted in 26.5 million reads per library. Differential expression was measured using the DESeq2 program [35 (link)]. TopHat (version 1.4.1) was used to align RNA-seq paired reads to GENCODE (version 9.0) [36 (link), 37 ]. Cufflinks (version 1.3.0) and BEDTools [38 (link), 39 (link)] were used to calculate raw counts for each GENCODE transcript. For this study X and Y chromosome transcripts were omitted.
10
Total RNA Purification and RT-qPCR Analysis
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Total tissue RNA was purified using QIAzol Lysis Reagent and the RNeasy kit (QIAGEN), and mRNA was isolated using the Dynabead mRNA Purification Kit (Thermo Fisher Scientific). For RT-PCR, cDNA was synthesized using Superscript IV (Thermo Fisher Scientific), and real-time PCR was performed using SYBR green fluorescence on a HT7900 thermal cycler (ABI) as previously described (Patino et al., 2006 (link)). The average cycle threshold (Ct) of the respective gene was normalized to housekeeping gene TIF (EIF3F) (see Table S2 for primer sequences). For RNA-Seq, mRNAs purified from 2 μg of iWAT total RNA were used to synthesize double-stranded cDNAs using the SuperScript Double-Stranded cDNA Synthesis Kit (Thermo Fisher Scientific).
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