RNA-Seq strand-specific libraries were constructed using a VAHTS Total RNA-Seq (H/M/R) Library Prep Kit (Vazyme, China). The original gene expression data set GSE75819 was downloaded from the GEO public database. We used R 4.0.0 to analyse the expression matrix. The robust multiarray average (RMA) method was used to pre-process data, including background adjustment, normalization, and log value conversion. The Limma package was used to search for differential genes. The threshold of up and down genes was set as |log FC| ≥ 1 and P ≤ 0.05.
Vahts total rna seq h m r library prep kit
Manufactured by Vazyme
Sourced in China
The VAHTS Total RNA-seq (H/M/R) Library Prep Kit is a laboratory equipment product designed for the preparation of RNA sequencing libraries. It is suitable for use with human, mouse, and rat samples.
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Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Novaseq 6000, by Illumina (4 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Novaseq 6000 platform, by Illumina (2 mentions)
Kapa stranded rna seq library prep kit, by Roche (2 mentions)
Vahtstm dna clean beads, by Illumina (2 mentions)
Trizol reagent kit, by Thermo Fisher Scientific (2 mentions)
Rneasy minelute cleanup kit, by Qiagen (2 mentions)
Vahtstm dna clean beads, by Vazyme (2 mentions)
Hiseq x ten, by Illumina (1 mentions)
Rneasy plus kit, by Qiagen (1 mentions)
Agilent 2100 biological, by Agilent Technologies (1 mentions)
Agilent 2200, by Agilent Technologies (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Ampure bead, by Beckman Coulter (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Rnase r, by Illumina (1 mentions)
Vahts rna clean beads, by Vazyme (1 mentions)
18 protocols using vahts total rna seq h m r library prep kit
1
RNA-seq Differential Gene Analysis
2
RNA-seq Library Prep and Analysis
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For RNA-seq experiments, polyA RNA-enriched and strand-specific libraries were constructed with the VAHTS Total RNA-seq (H/M/R) Library Prep Kit (Vazyme Biotech Co., Ltd), and the libraries were sequenced using the Illumina HiSeq X Ten system (Annoroad Gene Technology Corporation). We trimmed and mapped reads to the mouse mm9 reference assembly by the TopHat2 software [30 (link)] using default parameters except that we reduced maximum insertion and deletion length to 2 bp, and kept only uniquely mapped, “no mixed” and “no discordant” reads. For differential gene expression analysis, we analyzed raw read counts for GENCODE M1 genes using HTSeq [31 (link)] and then calculated statistics of differential expression via DESeq2 version 1.8.2 [32 (link)] with default parameters. To define differentially expressed genes, we used false discovery rate (FDR) 0.05 and log2 (fold change) > 1 or < − 1 as thresholds. We performed GO analysis using DAVID bioinformatics tools [33 (link)].
3
Transcriptomic Profiling of CTGF-Induced AEC2s
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Lineage tracing AEC2s were isolated from mice treated with or without CTGF administration. Total RNA was extracted using a RNeasy Plus Kit (Qiagen, Germany), and 1 μg of RNA was used as input material for the RNA sample preparations. RNA-seq strand-specific libraries were constructed using the VAHTS Total RNA seq (H/M/R) Library Prep Kit (Vazyme, China) according to the manufacturer’s instructions. Cluster was generated by cBot after the library was diluted to 10 pM and then sequenced on the Illumina NovaSeq 6000 platform (Illumina, USA). Library construction and sequencing were performed by Sinotech Genomics Co., Ltd. (Shanghai, China).
We performed a Gene Ontology (GO) analysis for biological processes, cellular components and molecular function and a KEGG pathway analysis (Kyoto Encyclopedia of Genes and Genomeshttp://www.genome.ad.jp/kegg ) via the enrich R package. We classified DEGs according to the official classification of KEGG annotation results and enriched the path function with Phyper. DIAMOND [19 (link)] was used to map the DEGs onto the STRING [20 (link)] database to determine the interaction between DEG-encoded proteins using homology with known proteins. For the whole interaction result, we provide an input file that can be directly imported into Cytoscape for network analysis.
We performed a Gene Ontology (GO) analysis for biological processes, cellular components and molecular function and a KEGG pathway analysis (Kyoto Encyclopedia of Genes and Genomes
4
Strand-specific RNA-seq Library Preparation
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The RNeasy mini kits were used to isolate the RNA, and strand-specific libraries were prepared using the VAHTS Total RNA-seq (H/M/R) Library Prep Kit (Vazyme, China). In Brief, RNA was purified after removal of rRNA by magnetic beads. The cleaved RNA fragments, which were segmented with divalent cations(94°C, 8 min), were used to synthesize the first strand cDNA; then, the reverse transcriptase and random primers were used to generate the second strand cDNA. After PCR purification and enrichment, the final cDNA library was generated. Then, Qubit® 2.0 Fluorometer (Life Technologies, USA) was used for quantification, and Agilent 2100 biological (Agilent Technologies, USA) was used for verification, so as to determine the size of the insert and calculate the molar concentration. cBot was used for cluster analysis, and Illumina NovaSeq 6000 (Illumina, USA) was sequenced. Shanghai Sinomics Corporation constructed and sequenced the library.
5
SNORA71A Overexpression mRNA Sequencing
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mRNA sequencing was operated in SNORA71A‐overexpressing MDA‐MB‐231 and negative control cells. Libraries were constructed via VAHTS™ Total RNA‐seq (H/M/R) Library Prep Kit (#NR603; Vazyme Biotech, Nanjing, China) following the user guides. In brief, mRNA was extracted using oligo‐dT magnetic beads and processed to mRNA fragments under divalent cations and high‐temperature conditions. Fist‐strand cDNA was synthesized by reverse transcriptase using random primers. The cDNA libraries were constructed via PCR and purified using Ampure Beads (Beckman, Brea, CA, USA). The quality of the DNA fragment was measured by Agilent 2200 (Agilent, Santa Rosa, CA, USA). Finally, RNA sequencing was performed using an Illumina HiSeq 2500 platform.
6
RNA-seq workflow for NRCFs
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Total RNA from NRCFs in the first part of the in vitro experiments was isolated using the RNeasy Mini Kit (Qiagen, Germany). After assessment of the integrity of total RNA using the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., USA), the samples with RNA integrity number > 7.0 were used for sequencing. RNA-sequencing strand-specific libraries were constructed using the VAHTS Total RNA-seq (H/M/R) Library Prep Kit (Vazyme, Nanjing, China). The library was then diluted to 10 pM, after which clusters were generated and sequenced on the Illumina NovaSeq 6000 platform (Illumina, USA). Differential expression analysis of mRNA was performed using the R package edgeR.
7
Liver RNA-Seq Library Preparation
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Total RNA from the sepsis model and sham-operated mouse liver tissues was extracted with TRIzol Reagent (Invitrogen, CA, USA). Further, ~3 μg total RNA from every sample was subjected to a VAHTS Total RNA-Seq (H/M/R) Library Prep Kit (Vazyme Biotech Co., Ltd, Nanjing, China) to eliminate ribosomal RNA and retain other classes of RNAs such as noncoding RNAs and mRNAs. We treated purified RNA employing 40 U RNase R (Epicenter, New England Biolabs, MA, USA) at 37°C for 3 h, followed by TRIzol purification. Our lab used a KAPA Stranded RNA-Seq Library Prep Kit (Roche, Basel, Switzerland) to prepare an RNA-Seq library, which was subjected to deep sequencing with Illumina HiSeq 4000 (CA, USA) at Aksomics, Inc. (Shanghai, China).
8
RNA Extraction and Sequencing from Mouse Kidney
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Total RNA was extracted from mouse kidney tissues using MiRNeasy Mini Kit (Cat#217004, Qiagen, Germany). Then, RNA was purified using VAHTS RNA Clean Beads (N412-01, Vazyme, CN), DNase I, and RNase-free (EN401, Vazyme, CN). Quality control (QC) was achieved using NanoDrop 2100 (Thermo Fisher Scientific, United States) and Agilent Bioanalyzer 4200 (Agilent Technologies, United States).
Small RNA (sRNA) (∼21 nucleotides) and whole transcriptome libraries were generated using the QIAseq miRNA Library Kit (Cat#331505, Qiagen, Germany) and VAHTS Total RNA-seq (H/M/R) Library Prep Kit (NR603-01, Vazyme, China) following the manufacturers’ instructions. All libraries were quantified using Agilent 2100 Bioanalyzer and Qubit® 3.0 fluorometer (Invitrogen; Thermo Fisher Scientific, Inc.). sRNA libraries and whole transcriptome libraries were sequenced on the Illumina Xten and Illumina NovaSeq 6000 (Illumina Inc., San Diego, CA, United States), respectively.
Small RNA (sRNA) (∼21 nucleotides) and whole transcriptome libraries were generated using the QIAseq miRNA Library Kit (Cat#331505, Qiagen, Germany) and VAHTS Total RNA-seq (H/M/R) Library Prep Kit (NR603-01, Vazyme, China) following the manufacturers’ instructions. All libraries were quantified using Agilent 2100 Bioanalyzer and Qubit® 3.0 fluorometer (Invitrogen; Thermo Fisher Scientific, Inc.). sRNA libraries and whole transcriptome libraries were sequenced on the Illumina Xten and Illumina NovaSeq 6000 (Illumina Inc., San Diego, CA, United States), respectively.
9
RNA-seq library preparation for ESCs and ICM
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Total RNAs were extracted as described above. For ESCs, two or three replicates for each sample were prepared, and 1 μg RNA per sample was subjected to rRNA elimination and RNA-seq library generation using VAHTS Total RNA-seq (H/M/R) Library Prep Kit (Vazyme) following manufacturer’s instructions. For ICM cells, two replicates for each sample were prepared, and purified RNAs were subjected to library generation using SMARTer Stranded Total RNA-Seq Kit (Takara Bio). Libraries were sequenced on the Illumina NovaSeq 6000 platform with paired ends and 150-bp read lengths at Berry Genomics.
10
RNA-seq Analysis of M1 and M0 Macrophages
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RNA sequencing (RNA-seq) of lipopolysaccharide (LPS)- and interferon-gamma (IFN-γ)-stimulated RAW 264.7 and control cells was performed using the Illumina NovaSeq 6000 platform (Illumina, USA) to identify lncRNAs with different expression levels in M1 and M0 macrophages. Total RNA was extracted using the TRIzol reagent (Invitrogen, USA). RNA integrity was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc., USA). Strand-specific libraries were constructed using the VAHTS total RNA-seq (H/M/R) library prep kit (NR306-01; Vazyme, China) according to the manufacturer's instructions, and StringTie was used to count the fragments within each gene (31 (link)). Finally, a differential expression analysis of mRNAs and lncRNAs was performed using the edgeR package of R.
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