Illumina-based mRNA-seq libraries were prepared from 1 μg RNA following previously published methods3 (link). mRNA was purified from total RNA using Sera-mag oligo(dT) magnetic beads (GE Healthcare Life Sciences) and then fragmented in the presence of divalent cations (Mg2+) at 94 °C for 6 min. The resulting fragmented mRNA was used for first-strand cDNA synthesis using random hexamers and reverse transcriptase, followed by second-strand cDNA synthesis using DNA polymerase I and RNaseH. Double-stranded cDNA was end-repaired using T4 DNA polymerase, T4 polynucleotide kinase and Klenow polymerase. The DNA fragments were then adenylated using Klenow exo-polymerase to allow the ligation of Illumina Truseq HT adapters (D501–D508 and D701–D712). All enzymes were purchased from Enzymatics. Following library preparation, quality control and quantification were performed using a 2100 Bioanalyzer instrument (Agilent) and the Quant-iT PicoGreen dsDNA Reagent (Invitrogen), respectively. Libraries were sequenced using Illumina HiSeq4000 sequencers to generate 50-bp single-end reads.
Sera mag oligo dt magnetic beads
Manufactured by GE Healthcare
Sourced in United Kingdom
Sera-mag oligo(dT) magnetic beads are a laboratory tool used for the isolation and purification of mRNA from biological samples. These beads contain oligo(dT) sequences that selectively bind to the poly(A) tail of mRNA molecules, allowing for their extraction from a complex mixture of nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 4000 sequencer, by Illumina (2 mentions)
Quant it picogreen dsdna reagent, by Thermo Fisher Scientific (2 mentions)
Truseq ht adapters, by Illumina (2 mentions)
2100 bioanalyzer instrument, by Agilent Technologies (2 mentions)
Hiseq 4000, by Illumina (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
5200 fragment analyser, by Agilent Technologies (1 mentions)
Kapa dual indexed adapter kit, by Roche (1 mentions)
Hiseq 2500 sequencer, by Illumina (1 mentions)
Truseq stranded mrna sample preparation kit, by Illumina (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Truseq small rna sample prep kit, by Illumina (1 mentions)
Rneasy protect cell mini kit, by Qiagen (1 mentions)
Hiseq 2000 system, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Qubit analyzer, by Thermo Fisher Scientific (1 mentions)
Oligotex direct mrna mini kit, by Qiagen (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500 sequencing system, by Illumina (1 mentions)
Nebnext mrna library prep reagent set, by New England Biolabs (1 mentions)
6 protocols using sera mag oligo dt magnetic beads
1
Illumina-based mRNA-seq Library Preparation
2
Illumina-based mRNA Sequencing Library Prep
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Illumina-based mRNA sequencing libraries were prepared from 1 μg RNA following Finkel and colleagues [61 (link)]. Briefly, mRNAs were selected using Sera-mag oligo(dT) magnetic beads (GE Healthcare Life Sciences). RNAs were washed and fragmented at 94°C for 6 minutes. First-strand cDNA synthesis was performed using random hexamers and reverse transcriptase (Superscript III reverse transcriptase, Invitrogen, Carlsbad, CA). Second-strand cDNA synthesis was done using DNA Polymerase I and RNAseH. Double-stranded cDNAs were end-repaired using T4 DNA polymerase, T4 polynucleotide kinase, and Klenow polymerase. The DNA fragments were then adenylated using Klenow exo-polymerase to allow the ligation of Illumina adapters (Kapa Dual-indexed adapter kit, Roche, Basel, Switzerland). Unless specified, reagents were purchased from Enzymatics. Library quality control and quantification were performed using the 5200 Fragment Analyser and the NGS fragment kit (Agilent Technologies, Santa Clara, CA). Libraries were sequenced using Illumina HiSeq4000 sequencers to generate 50-bp single-end reads.
3
Illumina-based mRNA-seq Library Preparation
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Illumina-based mRNA-seq libraries were prepared from 1 μg RNA following previously published methods3 (link). mRNA was purified from total RNA using Sera-mag oligo(dT) magnetic beads (GE Healthcare Life Sciences) and then fragmented in the presence of divalent cations (Mg2+) at 94 °C for 6 min. The resulting fragmented mRNA was used for first-strand cDNA synthesis using random hexamers and reverse transcriptase, followed by second-strand cDNA synthesis using DNA polymerase I and RNaseH. Double-stranded cDNA was end-repaired using T4 DNA polymerase, T4 polynucleotide kinase and Klenow polymerase. The DNA fragments were then adenylated using Klenow exo-polymerase to allow the ligation of Illumina Truseq HT adapters (D501–D508 and D701–D712). All enzymes were purchased from Enzymatics. Following library preparation, quality control and quantification were performed using a 2100 Bioanalyzer instrument (Agilent) and the Quant-iT PicoGreen dsDNA Reagent (Invitrogen), respectively. Libraries were sequenced using Illumina HiSeq4000 sequencers to generate 50-bp single-end reads.
4
Tetrahymena Transcriptome and Small RNA Profiling
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Total RNA was extracted from Tetrahymena cells using the RNeasy Protect Cell minikit (Qiagen), as described (TetraFGD, http://tfgd.ihb.ac.cn/index/smphelp ). Polyadenylated transcripts were enriched using Sera-Mag magnetic oligo-dT beads (GE). First strand-specific libraries were constructed using the Illumina TruSeq Stranded mRNA sample preparation kit (RS-122-2101). Small RNA was enriched by a mirVana™ miRNA isolation kit (Ambion). Small RNA libraries were constructed using the Illumina TruSeq Small RNA Sample Prep kit (RS-200-0012). Sequencing was performed using an Illumina HiSeq-2500 sequencer. Analysis of polyadenylated transcripts was performed as described previously (Xiong et al. 2012 (link); Feng et al. 2017 (link)). Analysis of small RNA was performed as described (Schoeberl et al. 2012 (link)).
5
Transcriptome Profiling of Leaf Tissues
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We constructed sequence libraries from three segments (LA, LM, and LB) of leaves generated from tip-burn susceptible and resistant lines and kale (Additional file 1 : Figure S1A). Total RNA was extracted from 100 mg of each tissue using Trizol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. To remove any DNA contamination, samples were treated using the Oligotex Direct mRNA Mini Kit (Qiagen, Hilden, Germany). The concentration of the mRNA was determined using a Qubit analyzer (Invitrogen, Carlsbad, CA, USA). The cDNA libraries were prepared according to the instructions of the manufacturer of the sequencing system (Illumina, San Diego, CA, USA). Poly(A) mRNA was purified using Sera-Mag Magnetic Oligo (dT) Beads (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) from 20 μg total RNA. The cDNA library products were sequenced on a paired-end flow cell using an Illumina HiSeq™ 2000 system.
6
Transcriptome Analysis of Algal Stress Response
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Total RNA (10 mg) extracted from each algal sample (0, 6, and 12 h of ND and 6 and 12 h of ND supplemented with 10 mM cerulenin; in triplicate) was treated by DNase I (Invitrogen; 18047019), and then mRNA was purified with Seramag Magnetic Oligo(dT) Beads (GE Healthcare; 38152103011150). The transcriptome libraries were constructed using the NEBNext mRNA Library Prep Reagent Set (New England Biolabs; E6100) and sequenced for two 150-bp runs (paired end) using a HiSeq 2500 sequencing system (Illumina) by Sangon Biotech. Reads were aligned to the C. zofingiensis genome (https://phytozomenext.jgi.doe.gov/info/Czofingiensis_v5_2_3_2) with TopHat (version 2.0.4; Trapnell et al., 2012) . The RNA-seq data can be accessed via the accession number GSE113802 in the Gene Expression Omnibus.
For each of the RNA-seq data sets, gene expression was measured as the numbers of aligned reads to annotated genes by Cufflinks (version 2.0.4) and normalized to fragments per kilobase million values. DEGs between two conditions were defined as followed: the fragments per kilobase million value of at least one condition was no less than 1 and gene expression showed at least a 2fold change with the false discovery rate-adjusted P , 0.05. The genes in each cluster were manually categorized into 11 groups according to Liu et al. (2019) .
For each of the RNA-seq data sets, gene expression was measured as the numbers of aligned reads to annotated genes by Cufflinks (version 2.0.4) and normalized to fragments per kilobase million values. DEGs between two conditions were defined as followed: the fragments per kilobase million value of at least one condition was no less than 1 and gene expression showed at least a 2fold change with the false discovery rate-adjusted P , 0.05. The genes in each cluster were manually categorized into 11 groups according to Liu et al. (2019) .
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