Libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA-Seq Kit (NEW ENGLAND BioLabs, Inc., UK). The isolation of mRNA was performed using the Poly(A) RNA Selection Kit (LEXOGEN, Inc., Austria). The isolated mRNAs were used for the cDNA synthesis and shearing, following manufacture's instruction. Indexing was performed using the Illumina indexes 1-12. The enrichment step was carried out using of PCR. Subsequently, libraries were checked using the Agilent 2100 bioanalyzer (DNA High Sensitivity Kit) to evaluate the mean fragment size. Quantification was performed using the library quantification kit using a StepOne Real-Time PCR System (Life Technologies, Inc., USA). High-throughput sequencing was performed as paired-end 100 sequencing using HiSeq X10 (Illumina, Inc., USA).
Poly a rna selection kit
Manufactured by Lexogen
Sourced in Austria
The Poly(A) RNA Selection Kit is a laboratory tool designed to selectively isolate and purify polyadenylated (poly(A)) RNA from total RNA samples. The kit utilizes oligo(dT) magnetic beads to capture and enrich for mRNA molecules, which typically contain a poly(A) tail. This process enables the separation of poly(A) RNA from other RNA species, such as ribosomal and non-polyadenylated RNA, facilitating downstream applications like gene expression analysis and RNA-sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Stepone real time pcr system, by Thermo Fisher Scientific (31 mentions)
Nebnext ultra 2 directional rna seq kit, by New England Biolabs (25 mentions)
Novaseq 6000, by Illumina (14 mentions)
Indexes 1 12, by Illumina (9 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (9 mentions)
Tapestation hs d1000 screen tape, by Agilent Technologies (9 mentions)
Smarter stranded rna seq kit, by Takara Bio (7 mentions)
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Hiseq x10, by Illumina (6 mentions)
Hiseq 2500, by Illumina (6 mentions)
Nd 2000 spectrophotometer, by Thermo Fisher Scientific (5 mentions)
Indices 1 12, by Illumina (4 mentions)
Hiseq 2500 system, by Illumina (2 mentions)
High sensitivity dna kit, by Agilent Technologies (2 mentions)
Agilent 2100, by Agilent Technologies (2 mentions)
Hiseq x10 system, by Illumina (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Thermo Fisher Scientific (1 mentions)
Rlt lysis buffer, by Qiagen (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
40 protocols using poly a rna selection kit
1
mRNA Library Preparation for RNA-Seq
2
Transcriptome Profiling by RNA-Seq
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Libraries were prepared from total RNA using a SMARTer Stranded RNA-Seq Kit (Clontech Laboratories, Inc., USA). mRNA was isolated with Poly (A) RNA Selection Kit (LEXOGEN, Inc., Austria); indexed with Illumina indices 1-12; and enriched with PCR. Libraries were assessed for mean fragment size using an Agilent 2100 Bioanalyzer (DNA High Sensitivity Kit), quantified with StepOne Real-Time PCR System (Life Technologies, Inc., USA). High-throughput sequencing was made as paired-end 100 sequencing with HiSeq 2500 system (Illumina, Inc., USA). FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) for quality control of raw sequencing data. Adapters, and low-quality reads (http://hannonlab.cshl.edu/fastx_toolkit/) and BBMap (https://sourceforge.net/projects/bbmap/ ), trimmed reads were mapped to reference with TopHat.55 Gene expression levels were estimated based on RC (read count) and FPKM (fragments per kb per million reads) values determined using BEDTools56 and Cufflinks.57 (link) then normalized with EdgeR within R (https://www.r-project.org ) Quantile normalization. Data mining and graphic visualized with ExDEGA (E-Biogen, Inc., Seoul, Republic of Korea). RNA-sequencing data have been stored at Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo accession no. GSE183220).
3
RNA-Seq Analysis of A549 Cells
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A TRIzol® reagent was used to extract total RNA from A549 cells. RNA concentrations and RNA purity were measured using NanoDropTM 2000 for conducting an RNA-seq analysis. The SMARTer Stranded RNA-Seq Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to prepare libraries from total RNA, and the poly(A) RNA Selection Kit (LEXOGEN, Inc., Vienna, Austria) was used to isolate mRNA. The cDNA synthesis and shearing were performed using the isolated mRNAs, following the manufacturer’s instruction and the Illumina indexes 1–12 were used to perform indexing. PCR was used to carry out the enrichment step and the Agilent 2100 bioanalyzer (DNA High Sensitivity Kit (Agilent, Santa Clara, CA, USA)) was used to check libraries for evaluating the mean fragment size. Quantification was performed on a StepOne Real-Time PCR System (Life Technologies, Inc., Carlsbad, CA, USA) using the library quantification kit. To perform high-throughput sequencing as paired-end 100 sequencing, HiSeq 2500 (Illumina, Inc., San Diego, CA, USA) was used and ebiogen (ebiogen, Seoul, Korea) conducted the RNA sequence analysis.
4
Isolation and Sequencing of hcSMC RNA
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hcSMCs at passage 7 were washed with cold PBS and then lysed in RLT lysis buffer (Qiagen). RNA was isolated using RNeasy kit (Qiagen), according to the manufacturers' protocol. RNA integrity was evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies). The RNA integrity numbers exceeded 8.5 in all samples. RNA was selected using Poly(A) RNA Selection Kit (Lexogen) and sequencing libraries prepared with Lexogen QuantSeq V2. DNA fragments of 200–800 bp for RNA-seq were selected. Cluster generation and sequencing was carried out by using the Illumina HiSeq 2500 system with a read length of 50 nucleotides (single-read). Sequence reads that passed the Illumina quality filtering were aligned to the human reference genome GRCh38 with star-aligner and gene annotation from ENSEMBL (GRCh38.95). Analysis of differential expression of mRNA was using the DeSeq2 software at default settings. This dataset was submitted to the European Nucleotide Archive (ENA) (https://www.ebi.ac.uk/ena/browser/home ) with the accession reference ERP127239 .
5
RNA-Seq Library Preparation and Sequencing
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Libraries were prepared using 2 μg of total RNA using a SMARTer Stranded RNA-Seq Kit (Clontech Laboratories, Inc., Redwood City, CA, USA). mRNA enrichment was performed using a Poly(A) RNA Selection Kit (Lexogen, Inc., Vienna, Austria). The isolated mRNA was used for cDNA synthesis and shearing following the manufacturer’s instructions. Indexing was performed using Illumina indexes 1–12. The enrichment step was carried out using polymerase chain reaction (PCR). Subsequently, the libraries were checked using an Agilent 2100 bioanalyzer and DNA High Sensitivity Ki t (5067-4626, Agilent Technologies, Inc., Santa Clara, CA, USA) to evaluate the mean fragment size. Quantification was performed using the library quantification kit for the StepOne Real-Time PCR System (Life Technologies, Inc., Carlsbad, CA, USA). High-throughput sequencing was performed as sequencing of paired-end 100 base pairs using a HiSeq 2500 (Illumina, Inc., San Diego, CA, USA).
6
Nanopore Direct cDNA Sequencing for Transcriptome
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Three biological replicates of the seedling and callus samples from the same tissue samples used for Illumina RNA-Seq were sequenced by the Nanopore direct cDNA sequencing protocol. Briefly, mRNA was first isolated from 10 ug total RNA with Poly(A) RNA Selection Kit (Lexogen), followed by direct cDNA library preparation with SQK-DCS109 kit (Oxford Nanopore). The protocol version for library preparation was DCS_9090_v109_revB_04Feb2019. The cDNA library was loaded onto a FLO-MIN106D R9 flowcell and sequenced on MinION (Oxford Nanopore).
FAST5 raw data was converted to FASTQ data using the basecaller Guppy version 3.4.5 (Oxford Nanopore) with default parameters. Two steps of trimming were employed. Adapter sequence was first trimmed by porechop (version 0.2.4) (https://github.com/rrwick/Porechop) with parameters "--check_reads 10000 --adapter_threshold 100 --end_size 100 --min_trim_size 5 --end_threshold 80 --extra_end_trim 1 --middle_threshold 100 --extra_middle_trim_good_side 5 --extra_middle_trim_bad_side 50", and then poly A was trimmed by the software cutadapt (version 2.6) 70 with the options of " -g T{12} -e 0.1 -a A{12} -n 100". Trimmed reads were aligned to A188Ref1 as unstranded spliced long reads using MiniMap2 (version 2.14) 71 with the parameter "ax splice". Merged alignments from three replicates were input to StingTie2 for generating assembled transcripts.
FAST5 raw data was converted to FASTQ data using the basecaller Guppy version 3.4.5 (Oxford Nanopore) with default parameters. Two steps of trimming were employed. Adapter sequence was first trimmed by porechop (version 0.2.4) (https://github.com/rrwick/Porechop) with parameters "--check_reads 10000 --adapter_threshold 100 --end_size 100 --min_trim_size 5 --end_threshold 80 --extra_end_trim 1 --middle_threshold 100 --extra_middle_trim_good_side 5 --extra_middle_trim_bad_side 50", and then poly A was trimmed by the software cutadapt (version 2.6) 70 with the options of " -g T{12} -e 0.1 -a A{12} -n 100". Trimmed reads were aligned to A188Ref1 as unstranded spliced long reads using MiniMap2 (version 2.14) 71 with the parameter "ax splice". Merged alignments from three replicates were input to StingTie2 for generating assembled transcripts.
7
RNA-seq Library Preparation and Analysis
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Total RNA was isolated using TRIzol reagent (Invitrogen, 15596018), and RNA quality and quantification were performed using an Agilent 2100 bioanalyzer (Agilent Technologies) and an ND-2000 Spectrophotometer (Thermo). Library preparation was performed using the NEBNext Ultra II Directional RNA-Seq Kit (NEW ENGLAND BioLabs, E7760L). Using a Poly(A) RNA Selection Kit, mRNA was extracted (LEXOGEN, 157.96) and used according to the manufacturer’s instructions for cDNA synthesis and shearing. Indexing was performed using Illumina indices 1–12 and was enriched by PCR. The mean fragment sizes were evaluated, and libraries were checked using a TapeStation HS D1000 Screen Tape (Agilent Technologies). Quantification was performed using the StepOne Real-Time PCR System (Life Technologies), and high-throughput sequencing was performed using NovaSeq 6000 (Illumina). FastQC was used to control the quality of the raw sequencing data, and adapters and low-quality reads (
8
RNA-Seq Library Preparation and Sequencing
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Total RNA was isolated using TRIzol reagent (Invitrogen). RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies), and the RNA was quantified using an ND-2000 Spectrophotometer (Thermo). Libraries were prepared from the total RNA using the NEBNext Ultra II Directional RNA-Seq Kit (New England BioLabs). mRNA was isolated using the Poly(A) RNA Selection Kit (Lexogen) and was used for cDNA synthesis and shearing, following the manufacturer’s instructions. Indexing was performed using Illumina indices 1–12. The libraries were enriched using PCR. Fragment size was evaluated using the Agilent 2100 Bioanalyzer (DNA High Sensitivity Kit). Quantification was performed using a library quantification kit and a StepOne RT‒PCR System (Life Technologies). High-throughput sequencing was performed as paired-end 100 sequencing using a NovaSeq 6000 instrument (Illumina).
9
Comprehensive RNA-Seq Protocol for Transcriptome Analysis
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Total RNA was isolated and underwent quality assessment with Agilent 2000 bioanalyzer (Agilent Technologies, Netherlands). The library was generated with NEBNext Ultra II Directional RNA-Seq Kit (New England Biolabs, UK). Next, the mRNA was isolated, fragmented and synthesized using the Poly(A) RNA Selection Kit (Lexogen, Austria) according to the manufacturer’s instructions. The Illumina indexes 1-12 were used for indexing. The raw sequencing data underwent quality control by FastQC(https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). HiSeq X 10 (Illumina, USA) was utilized for high-throughput paired-end 100 sequencing, and the adapter and low-quality reads (< Q20) were excluded by FASTX_Trimmer ((http://hannonlab.cshl.edu/fastx_toolkit/ ) and BBMap ((https://sourceforge.net/projects/bbmap ). For analysis, the reads were mapped to the reference genome via TopHat [23 (link)]and the expression levels were calculated based on the Fragments Per Kilobase Million (FPKM) mapped reads from Cufflinks [24 (link)]. Quantile normalization was performed via EdgeR (R Development Core Team, 2016) and ExDEGA (E- biogen, Inc., Korea) was used for mining and graphic visualization of the data. A more detailed description of the method is provided in our previous study [4 (link)].
10
RNA-Seq Library Preparation Protocol
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Libraries were prepared from total RNA by using the NEBNext Ultra II Directional RNA-Seq Kit (NEW ENGLAND BioLabs, UK). Poly(A)-tailed mRNAs were isolated using a Poly(A) RNA Selection Kit (LEXOGEN, Austria). The isolated mRNAs were used for the synthesis of cDNA, which was then sheared, following the manufacturer’s instructions. Indexing was performed using the Illumina indices 1-12 (Illumina, USA). The enrichment step was carried out using polymerase chain reaction (PCR). Subsequently, libraries were checked using the Agilent 2100 bioanalyzer (DNA High Sensitivity Kit) to evaluate the mean fragment size. Quantification was performed using the library quantification kit using a StepOne Real-Time PCR System (Life Technologies, USA). High-throughput sequencing (paired-end 100 bp) was performed using HiSeq ×10 (Illumina).
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