After the total RNA extraction, mRNA was purified from the 20 μg of RNA using Sera-mag Magnetic Oligo (dT) Beads (Thermo Fisher Scientific, USA). Then the purified mRNA was broken into small pieces using fragmentation buffer under elevated temperature. These short fragments as templates were used to synthesize first strand cDNA. Subsequently, the second-strand cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, USA). The short cDNA fragments were purified with Qia-Quick PCR extraction kit and end-repair with EB buffer and ligation of A-tailing. Next, suitable fragments were selected as templates for PCR amplification to create the final cDNA library. Finally, after validating on an Agilent Technologie 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System, the cDNA library was sequenced at the Beijing Genomics Institute (BGI, Shenzhen, China) using Illumina HiSeq™ 2000 sequencing platform. Image data outputs from sequencing machine were transformed by base calling into sequence data, which is called raw reads.
Sera mag magnetic oligo dt beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Sera-mag Magnetic Oligo (dT) Beads are laboratory equipment designed for the purification and isolation of poly(A) messenger RNA (mRNA) from total RNA samples. The beads are coated with oligo(dT) sequences that selectively bind to the poly(A) tails of mRNA molecules, allowing for their capture and separation from the sample.
Automatically generated - may contain errors
Lab products found in correlation
Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Hiseq 2000, by Illumina (2 mentions)
Paired end sample prep kit, by Illumina (2 mentions)
Hiseq 2000 platform, by Illumina (2 mentions)
Hiseq 2000 sequencing platform, by Illumina (1 mentions)
Rna fragmentation reagent, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Tiangen Biotech (1 mentions)
Fragmentation solution, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Qubit 2.0 fluorometer, by Agilent Technologies (1 mentions)
Hiseq x ten platform, by Illumina (1 mentions)
Pcr purification kit, by Thermo Fisher Scientific (1 mentions)
Second strand buffer, by Thermo Fisher Scientific (1 mentions)
6 protocols using sera mag magnetic oligo dt beads
1
Illumina RNA-seq Library Preparation
2
Transcriptome Analysis of Hainan Beech
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A C. hainanensis leaf transcriptome library was constructed using an mRNA-seq assay for paired-end Illumina sequencing, which was performed at Majorbio Biopharm Technology Co., Ltd. (Shanghai, China). Poly(A) mRNA was isolated from total RNA by using Sera-mag Magnetic Oligo (dT) Beads (Thermo Fisher Scientific, USA), and then mRNA-enriched RNAs were chemically fragmented to short pieces using the RNA Fragmentation Reagent (Ambion, USA). Double-stranded cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). Subsequently, the Illumina Paired End Sample Prep kit (Illumina, USA) was used to construct a RNA-seq library which then was sequenced by Illumina HiSeq 2000 (Illumina, San Diego, CA).
3
Transcriptome Profiling of Radish Root under Chromium Stress
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Total RNA was extracted from root samples using TRIzol reagents (Tiangen Biotech Co., Ltd., China) according to the manufacturer's instructions. The RNAs were treated with RNase-free DNase I to eliminate contaminated genomic DNA. Two radish cDNA libraries, CK and Cr600, were constructed from control and 600 mg L−1 K2Cr2O7 treated root samples using the Illumina Paired End Sample Prep Kit. Briefly, poly (A) mRNA was enriched from total RNA using Sera-mag Magnetic Oligo (dT) Beads (Thermo Fisher Scientific, USA) and then mRNA-enriched RNAs were chemically fragmented to short pieces using the fragmentation solution (Ambion, USA). Double-stranded cDNA was generated using the Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen, USA). After that, the Illumina Paired End Sample Prep Kit was used for RNA-seq library construction and was then sequenced using Illumina HiSeq™ 2000.
4
Transcriptome Profiling of Biopsy Samples
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A total of 10 g RNA was extracted from the frozen biopsy tissue of each patient, then poly(A)+ mRNA was isolated using Sera-mag Magnetic Oligo(dT) Beads (Thermo, part # 1004815). Afterwards, the purified RNA was broken into short segments in Fragment buffer. Next, reverse transcription was performed to construct a cDNA library. RNA concentration was measured with a Qubit® 2.0 Fluorometer and RNA integrity number (RIN) was measured by use of a Bioanalyzer 2100 (Agilent, CA, USA). Sequencing was performed on the HiSeq2000 platform (Illumina Inc., San Diego, CA) and single-end reads (50 bp in length) were generated.
5
Pear mRNA-seq Transcriptome Profiling
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Poly (A) mRNA was enriched from 20 μg of total RNA from the NaCl-treated pear samples, using Sera-mag Magnetic Oligo (dT) beads (Thermo Fisher Scientific, USA). To avoid priming bias when synthesizing cDNA, the purified mRNA was fragmented to small pieces (~200 bp) using 1× fragmentation solution (Ambion, USA) at 94 °C for 5 min. These short mRNA fragments were used as templates, and random hexamer-primers were used as primers for first-strand cDNA synthesis. Double-stranded cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, USA). After synthesis of the second strand of cDNA, cDNA fragments went through an end repair and poly-A addition process, followed by ligation of sequencing adapters. The suitable fragment products were purified using agarose gel electrophoresis and enriched by PCR amplification to create a final library. The mixed cDNA library named ‘PDX’ was constructed using an mRNA-seq assay for paired-end transcriptome sequencing, which was performed by the Beijing Genomics Institute (Shenzhen, China) using an Illumina HiSeq™ 2000 platform, and 90 bp paired-end reads were generated. The sequencing data were deposited in the NCBI Sequence Read Archive (SRA, http://www.ncbi.nlm.nih.gov/sra/ ) as accession number SRX525736.
6
Paired-End RNA-Seq Library Preparation
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The cDNA libraries were constructed and sequenced by OriGene Technologies, Beijing, China. Specifically, mRNA was isolated from two total RNA samples (Grabherr et al., 2011) (link). To construct the paired-end RNA-seq libraries for the transcriptome analysis, 10 µg total poly(A) mRNA was isolated with Seramag Magnetic Oligo(dT) Beads (Thermo) and then fragmented in fragmentation buffer. The resulting fragments were used as templates for the first-strand cDNA synthesis with a random hexamer primer. The second cDNA strand was synthesized in 20 µl second strand buffer (Invitrogen) supplemented with 10 mM dNTP mix, 5 U/µL RNase H, and 10 U/µL DNA polymerase I. Short fragments were purified with the QIAquick PCR purification kit and resuspended in EB buffer for the endrepair step and addition of a poly(A) tail. Sequencing adapters were then added to the short fragments. After an agarose gel electrophoresis, suitable fragments were selected as templates for a PCR amplification. Finally, paired-end RNA-seq libraries were prepared as recommended by Illumina and then sequenced on the HiSeq TM X Ten platform (Illumina). The Illumina instrument software was used for data analysis and base calling.
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