Total RNA was isolated using TRIzol (Life Technologies, Carlsbad, CA). After total RNA was extracted, rRNA was removed by using VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina (Vazyme Biotech Co., Ltd, Nanjing, China) to retain other types of RNA, including mRNAs and ncRNAs. The enriched mRNAs and ncRNAs were fragmented into short fragments in fragmentation buffer (MgCl2) integrated into VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina (Vazyme Biotech Co., Ltd, Nanjing, China), then the RNA fragments were reverse-transcribed into cDNA with random primers. Second-strand cDNA was synthesized with DNA polymerase I, RNase H, dNTPs (dUTP instead of dTTP), and buffer. Next, the cDNA fragments were purified with a QiaQuick PCR extraction kit, end-repaired, underwent poly(A) addition, and ligated to Illumina sequencing adapters. Then, UNG (uracil-N-glycosylase) was used to digest the second-strand cDNA. The digested products were size-selected via agarose gel electrophoresis, PCR-amplified, and sequenced using an Illumina HiSeqTM 4000 by Gene Denovo Biotechnology Co. (Guangzhou, China).
Vahts total rna seq h m r library prep kit for
Manufactured by Illumina
Sourced in China
The VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina is a library preparation kit designed for whole transcriptome sequencing of human, mouse, and rat samples. The kit enables the conversion of total RNA into a library of template molecules suitable for subsequent cluster generation and DNA sequencing on Illumina platforms.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Hiseq 2500, by Illumina (3 mentions)
Rnase r, by Illumina (3 mentions)
Ribo off rrna depletion kit, by Vazyme (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Hiseq x10 platform, by Illumina (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
Rnaclean xp kit, by Beckman Coulter (2 mentions)
Rnase free dnase set, by Qiagen (2 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop nd 2000, by Thermo Fisher Scientific (1 mentions)
Hiseqtm 2500, by Illumina (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Dna clean beads, by Illumina (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Hiseq x ten reagent kit, by Illumina (1 mentions)
Epicenter ribo zero rrna removal kit, by Illumina (1 mentions)
Hiseq x ten system, by Illumina (1 mentions)
20 protocols using vahts total rna seq h m r library prep kit for
1
Total RNA Extraction and Sequencing
2
Transcriptomic Analysis of Papillary Thyroid Carcinoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TriZol reagent (Cat No. 15596018, Life Technologies, Carlsbad, CA, USA) was employed to isolate total RNA from the tissues as described by the manufacturer. RNA quality was checked through determination of 260/280 OD values on a NanoDrop ND-2000 instrument (Thermo Fisher Scientific, Waltham, MA, USA). Total RNAs of four paired PTC and neighboring non-malignant tissues were treated with VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina as described in the manufacturer provided manual. Subsequently, sequencing of the products was run on the Illumina HiSeqTM 2500 platform by Gene Denovo Biotechnology Co. (Guangzhou, China). The processed clean reads were then aligned to the human genome (version: hg38_GRCh38).
3
Transcriptome Analysis of Mouse Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA sequencing libraries were constructed according to the instructions of the manufacturer for the VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina (Vazyme, China). The libraries were sequenced on the Illumina Nova-seq 6000 platform. The raw sequenced reads were mapped to the mouse genome (mm10) using the STAR software (Dobin et al., 2013 (link)). Genes were annotated using the Ensemble database. The expression of the genes was quantified using HTseq (Anders et al., 2015 (link)), and the differentially expressed genes (DEGs) were identified using DEseq2 (Love et al., 2014 (link)). Genes with FDR < 0.01 and | Log2 (fold change)| > 1 were considered DEGs. Gene ontology (GO) analysis was performed using the DAVID database (Huang da et al., 2009 (link)). For the ChIRP-seq analysis, raw reads were mapped to the mouse genome (mm10) using the bowtie2 software (Langmead and Salzberg, 2012 (link)). Peaks were called with the MACS suite (Zhang et al., 2008 (link)). Motif enrichment analysis was performed using the MEME suite (Bailey et al., 2015 (link)).
4
Profiling Circular RNAs by RNA-seq
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following extraction, total RNA was treated with RNase R to degrade the linear RNA and purified with RNeasy MinElute Cleanup Kit (Qiagen, Inc., Valencia, CA, USA). Next, a strand-specific library was constructed with VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina according to the manufacturer's protocol. In brief, ribosomal RNA was removed to retain the circRNAs. The enriched circRNAs were broken into short fragments using a fragmentation buffer and reverse transcribed into cDNA with random primers. Secondly, strand cDNA fragments synthesized by DNA polymerase I were purified with VAHTSTM DNA Clean Beads and liquated to Illumina sequencing adapters. Uracil-N-glycosylase was used to digest the second-strand cDNA. The digested products were purified with VAHTSTM DNA Clean Beads, amplified, and sequenced with Illumina HiSeq™ 2500 by Gene Denovo Biotechnology Co. (Guangzhou, China). The edgeR package (http://www.rproject.org/ ) was used to identify differentially expressed circRNAs. Some significant circRNAs were blasted against the circBase for annotation [13 (link)]. The circRNAs that could not be annotated were defined as novel circRNAs.
5
Comprehensive RNA-seq Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 2 µg of RNA sample from each subject was prepared as the input for RNA-seq. In the first step, ribosomal RNA (rRNA) was removed using Epicenter Ribozero™ rRNA Removal kit (Epicenter; Illumina, Inc.) and rRNA-free components were cleaned by ethanol precipitation. Next, the RNAs were submitted for library preparation according to the method described in a previous study (29 (link)). It was prepared using the VAHTS Total RNA-seq (H/M/R) Library Prep kit for Illumina (cat. no. NR603-01; Vazyme Biotech Co., Ltd.) according to the manufacturer's protocols. In brief, the following steps, including fragmentation, reverse transcription, adaptor ligation and preamplification, were performed sequentially. Finally, the libraries were purified, quality-tested and quantified using the Agilent Bioanalyzer 2100 system (Agilent Technologies, Inc.). The libraries were sequenced on Illumina HiSeq X Ten System by using HiSeq X Ten Reagent Kit (cat. no. FC-501-2501) with 2x150 bp paired-end technology.
6
RNA Isolation and Sequencing from HUVECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from HUVECs and purified using TRIzol® (cat. no. 15596026, Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions, the quantity and quality of the RNA were determined using an Agilent 4200 Bioanalyzer (Thermo, Fisher Scientific, Inc.). The RNA integrity was assessed by electrophoresis with denaturing agarose gels. Libraries were constructed using a VAHTS Total RNA-Seq(H/M/R) Library PrepKit for Illumina (cat. no. NR603-02; Vazyme Biotech Co., Ltd.) according to the manufacturer's protocol, and libraries were identified using a Qubit™ dsDNA HS Assay Kit (cat. no. Q32854; Invitrogen; Thermo Fisher Scientific, Inc.) and Agilent High Sensitivity DNA Kit (cat. no. 5067-4626; Agilent Technologies, Inc.). The qualities of the libraries were assessed using a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Inc.) and the concentration of DNA in the libraries was analyzed using an Agilent 2100 bioanalyzer (Agilent Technologies, Inc.). Clusters were generated bycBot (Illumina, Inc.) and the libraries were diluted to 10 pm, then sequencing was performed on the Illumina HiSeq 2500 according to the manufacturer's protocol at Shanghai Ao-Ji Biotechnology Co., Ltd (2x150 bp, paired-end). Details of the experimental procedures and instruments used are described in our previous study (24 (link)).
7
Illumina Library Prep and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sequencing libraries were prepared following manufacturer recommendations from the VAHTS™ Total RNA-seq (H/M/R) Library Prep Kit for Illumina®. The details of library construction were patented by the company (Vazyme, China). After cluster generation, the libraries were sequenced on an Illumina Hiseq X10 platform, and 150-bp paired-end reads were generated.
Raw reads in fastq format were first processed using in-house perl scripts. Clean reads were obtained by removing reads with adapters, reads in which unknown bases were more than 5% and low quality reads (if the percentage of low quality bases was greater than 50% in a given read, we defined the low quality base to be the base whose sequencing quality was no more than 10). At the same time, Q20, Q30, and GC contents were calculated for the clean reads. All downstream analyses were based on the clean reads.
The reference genome and gene model annotation files were downloaded directly from the genome website. The reference genome index was built using Bowtie (v2.1.0) [27 (link)], and the paired-end clean reads were aligned to the reference genome using TopHat (v2.1.1) [28 (link)].
Raw reads in fastq format were first processed using in-house perl scripts. Clean reads were obtained by removing reads with adapters, reads in which unknown bases were more than 5% and low quality reads (if the percentage of low quality bases was greater than 50% in a given read, we defined the low quality base to be the base whose sequencing quality was no more than 10). At the same time, Q20, Q30, and GC contents were calculated for the clean reads. All downstream analyses were based on the clean reads.
The reference genome and gene model annotation files were downloaded directly from the genome website. The reference genome index was built using Bowtie (v2.1.0) [27 (link)], and the paired-end clean reads were aligned to the reference genome using TopHat (v2.1.1) [28 (link)].
8
Comprehensive RNA Sequencing Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We extracted total RNA from two pairs of BC and adjacent noncancerous tissues or T24 cells with or without small interfering RNA targeting circRGNEF (si-circRGNEF) via TRIzol reagent (Invitrogen, Carlsbad, CA, USA). We subjected nearly 3 μg of total RNA from every sample to VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina (Vazyme Biotech Co., Ltd, Nanjing, China) to eliminate ribosomal RNA. We retained other RNA such as non-coding RNA and mRNA. We treated purified RNA with RNase R (Epicenter, 40 U, 37°C for 3 h), and then performed TRIzol purification. We prepared RNA-Seq libraries through the KAPA Stranded RNA-Seq Library Prep Kit (Roche, Basel, Switzerland), which were subjected to deep sequencing through Illumina HiSeq 4000 at Aksomics, Inc., Shanghai, China (accession number: H1712024).
9
Transcriptome Profiling of Gastric Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Firstly, we extracted total RNA from GC tissues and matched normal tissues by Trizol® Reagent (Invitrogen, USA). In total, 1 μg RNA was treated with Ribo-off rRNA Depletion Kit (Vazyme) before constructing the RNA-seq libraries. VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina (Vazyme) were utilized to prepare RNA-seq libraries following the manufacturer’s instructions. Furthermore, ribosome depleted RNA samples (~100 ng) were fragmented and then used for first- and second-strand cDNA synthesis with random hexamer primers. The ends of the cDNA fragments were repaired by DNA End Repair Kit. Then the cDNA fragments were modified with Klenow to add an A at the 3’ end of the DNA fragments, and finally ligated to adapters. We subjected purified dsDNA to 12 cycles of PCR amplification, and sequenced the libraries by Illumina sequencing platform on a 150 bp paired-end run. Sequencing reads from RNA-seq data were aligned using the spliced read aligner HISAT2, which was supplied with the Ensembl human genome assembly (Genome Reference Consortium GRCh38) as the reference genome. Then “combat-seq” R package was used to remove batch effects. Gene expression levels were calculated by the TPM (Transcripts Per Million). We utilized the GENCODE (v25) database to get annotations of mRNA in the human genome.
10
Circular RNA, miRNA, and mRNA Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A circRNA library, a small RNA library, and a mRNA library were constructed for the identification of circRNA, miRNA, and mRNA, respectively. To prepare for circRNA sequencing, linear RNAs were removed using RNase R (RNR07250, Epicentre) (1 unit/ μg) for 20 min treatment at 37°C. Ribosomal RNA (rRNA) was depleted in the total RNA using a Ribo-Zero Gold Kit (Epicentre) following the manufacturer’s instructions. After purification, the rRNA-depleted RNA products were fragmented using VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina (Vazyme Biotech Co., Ltd, Nanjing, Jiangsu, China). Three cDNA libraries were sequenced on Illumina Hiseq X-Ten platform (HaploX Biotechnology, Jiangxi, China) and 2×150 bp paired-end (PE150) reads were obtained using the HiSeq Control Software (HD3.5.0). Next, the sequencing reads were conducted for real-time sequencing image analysis and base-calling using Real-Time Analysis (v2.7.7). All Illumina sequencing raw and processed data were submitted to the GEO database (http://www.ncbi.nlm.nih.gov/geo/ ) under the accession number GSE139516.
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!