A total of 1 mg RNA samples were treated with VAHTS mRNA capture beads (Vazyme, China) to enrich polyA+ RNAs prior to constructing the RNA-seq libraries. The VAHTS mRNA-seq v2 library preparation kit of the Illumina Xten system (Vazyme, Nanjing, China) was used to prepare the RNA-seq libraries according to the manufacturer’s instructions. Briefly, polyA+ RNA samples (~100 ng) were fragmented and reverse transcribed into double strand cDNA. Then these cDNA fragments went through end repair, the addition of adenine tails, and ligation of the adaptors processes. The purified products were subjected to 12 cycles of PCR amplification to create the final cDNA libraries. These libraries were sequenced on 150 bp paired-end Illumina sequencing run. Sequenced readings were aligned using HISAT2 with human genome GRCh38 as a reference genome. Gene expression levels were calculated from fragments per kilobase of transcript per million mapped reads (FPKM). Gene ontology (GO) analysis was performed using the Metascape tool, and the given input list contained genes that were expressed at a lower level than the shENSA group (fold change (FC) < 0.8). GSEA was performed using GSEA software (v3.0) and molecular signature database (v7.0).
Vahts mrna capture beads
Manufactured by Vazyme
Sourced in China
VAHTS mRNA Capture Beads are magnetic beads designed for the efficient capture and purification of polyadenylated 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 the isolation of mRNA from complex RNA mixtures.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Hiseq 3000, by Illumina (5 mentions)
Novaseq 6000 system, by Illumina (5 mentions)
Rq1 dnase, by Promega (4 mentions)
Vahts mrna seq v2 library prep kit, by Illumina (3 mentions)
Nebnext ultra rna library prep kit, by New England Biolabs (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Novaseq 6000, by Illumina (2 mentions)
Hiscript 2 q select rt supermix, by Vazyme (2 mentions)
Ez rna methylation kit, by Zymo Research (2 mentions)
Kapa stranded mrna seq kit for platforms, by Illumina (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Vahts dna clean beads, by Vazyme (2 mentions)
Protoscript 2 buffer, by New England Biolabs (1 mentions)
Next ultra rna library prep kit, by Illumina (1 mentions)
Quick cip, by New England Biolabs (1 mentions)
First strand synthesis reaction buffer, by New England Biolabs (1 mentions)
Rna clean concentrator kit, by Zymo Research (1 mentions)
Smartspec plus, by Bio-Rad (1 mentions)
35 protocols using vahts mrna capture beads
1
RNA-seq Library Preparation and Analysis
2
mRNA Capture Using VAHTS Beads
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mRNA was captured by VAHTS mRNA Capture Beads (Vazyme, #N401,Nanjing, China) according to the manufacturer’s instructions.
3
RNA-seq library preparation from A549 cells
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Total RNA was extracted from A549 cells of different treatment groups using TRIzol and quantified using a NanoDrop (Thermo). RNA samples (1 μg) were treated with VAHTS mRNA capture beads (Vazyme, China) to enrich poly A+ RNA. The VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme, China) was used to prepare the RNA-seq libraries according to the manufacturer’s instructions. Briefly, approximately 100 ng of poly A+ RNA samples were fragmented and reverse transcribed into double-stranded cDNA. Then, these cDNA fragments were subjected to end repair, adenine tail addition, and adaptor ligation. The purified products were subjected to 12 cycles of PCR amplification to generate the final cDNA libraries. All library sizes and concentrations were confirmed on the Agilent 4200 TapeStation System and a Qubit 3.0 Fluorometer (Thermo). Finally, the libraries were sequenced on an Illumina NovaSeq 6000 by Haplox Biotechnology Co (Shenzhen, China).
4
RNA-Seq Analysis of SW480 Cells
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Total RNA (1 μg) was isolated from SW480 cells and treated with VAHTS mRNA Capture Beads (Vazyme, Nanjing, China) to enrich polyA+ RNA before constructing the RNA libraries. RNA library preparation was performed by using a VAHTS mRNA-sequencing v2 Library Prep Kit for Illumina (Vazyme, Nanjing, China). Paired-end sequencing was performed with an Illumina HiSeq 3000 at RiboBio Co., Ltd. (Guangzhou, China). For computational analysis of RNA-sequencing data, sequencing reads were aligned using the spliced read aligner HISAT2, which was supplied with the Ensemble human genome assembly (Genome Reference Consortium GRCh38) as the reference genome. Gene expression levels were calculated by fragments per kilobase of transcript per million mapped reads (FPKM). Gene set enrichment analysis (GSEA), a bioinformatic method used to assess whether sets of genes are significantly different, was performed. The method was used to compute the similarity between a query gene set compared to the gene sets available in the GSEA database and derived from published studies. The Java GSEA Desktop Application (http://www.broadinstitute.org/gsea/index.jsp ) was used with the hallmark gene set collections.
5
RNA-Seq Analysis of Ovarian Cancer Cells and Organoids
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RNA-sequencing assay was performed as previously described 47 (link). Briefly, a minimum of 1 mg of total RNA was isolated from ovarian cancer cells or organoids and treated with VAHTS mRNA Capture Beads (Vazyme) to enrich polyAþ RNA before constructing RNA libraries. RNA library preparation was performed by using VAHTS mRNA-seq v2 Library Prep Kit from Illumina (Vazyme). Paired-end sequencing was performed with Illumina HiSeq 3000 at RiboBio Co., Ltd. For computational analysis of RNA-sequencing data, sequencing reads were aligned using the spliced read aligner HISAT2, which was supplied with the Ensemble Human Genome Assembly (Genome Reference Consortium GRCh38) as the reference genome. The gene expression levels for each transcript were estimated as the number of reads per kilobase of exon model per million mapped reads (RPKM). Gene Set Enrichment Analysis (GSEA) was used for gene functional annotation. A gene is considered significantly differentially expressed if its expression differs between any two samples with the fold change > 2 and the p value < 0.05 as calculated by Cufflinks.
6
RNA-seq Analysis of SHEP MYCN-ER Cells
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Total RNA was extracted from 50 million SHEP MYCN-ER cells spiked-in with 15% Drosophila S2 cells using TRIzol reagent (Thermo Fisher Scientific, #15596018). mRNA was purified from 1 μg of total RNA with the VAHTS mRNA capture beads (Vazyme, #N401-02) and fragmented to 200 to 500 bp size long in 2× ProtoScript II buffer (New England Biolabs, #M0368). First- and second-strand DNA synthesis and adaptor ligation were conducted using an New England Biolabs Next Ultra RNA library prep kit for Illumina. The resulting strand-specific RNA-seq libraries were subjected to 150-bp paired-end sequencing on a NovaSeq 6000. All raw fastq reads were aligned to the Drosophila genome (dm6) and human genome (hg38) by HISAT2 version 2.1.0, respectively. Raw read counts were normalized to RPM (reads per million) per sample and then displayed in the UCSC genome browser as bigWig-formatted coverage tracks. Read counts at indicated regions were calculated by HTSeq-count (version 0.12.3) and were provided to DEseq2 (version 1.32.0) for differential gene expression analysis. Human gene expression was normalized using Drosophila spiked-in genes. FDR < 0.05 and log2FC >0.5 (or < −0.5) were used to identify differentially expressed genes.
7
DSCAM-AS1 Knockdown in MCF-7 Cells
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MCF‐7 cells were plated onto 6‐well plates and transfected with si‐NC, si‐DSCAM‐AS1‐1, or si‐DSCAM‐AS1‐2 the following day. Forty‐eight hours after transfection, total RNA was extracted. RNA integrity was validated using denaturing gel electrophoresis and ethidium bromide (EtBr) staining. The total RNA samples (1 μg) were treated with VAHTS mRNA Capture Beads (Vazyme, Nanjing, China; N401‐01) to enrich polyA+ RNA. RNA‐seq libraries were prepared using a VAHTS mRNA‐seq v2 Library Prep Kit for Illumina (Vazyme, Nanjing, China; NR601‐01) according to the manufacturer's instructions. The libraries were sequenced by an Illumina sequencing platform on a 150 bp paired‐end run. Sequencing reads were aligned using the spliced read aligner HISAT2. The Ensembl human genome assembly (Genome Reference Consortium GRCh38) was used as the reference genome.
8
Enrichment and Analysis of 5'Capped RNA
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PolyA-RNA was purified and enriched from 25 μg HepG2 total RNA by using VAHTS mRNA Capture Beads (Vazyme, N401-01). Enriched polyA-RNA were fragmented with NEBNext First Strand Synthesis Reaction Buffer (5 ×) in 15 μl volume at 94 °C for 9 min. Then the fragments were purified with 2 volumes of VAHTS RNA Clean beads. To enrich the 5’capped fragments, the fragmented RNA was incubated with 2 U Terminator exonuclease (Lucigen, TER51020), 1x Terminator reaction buffer A, and 20 U RRI in 40 μl volumes for 30 °C, 1 h. The RNA was purified by using RNA Clean & Concentrator Kits (ZYMO research, R1013). The terminator-treated RNA was treated with 5 μl Quick CIP (NEB, M0525S) and 1x CutSmart buffer at 37 °C for 30 min, followed by purification by RNA Clean & Concentrator Kits. Then, the eluted RNA was mixed with Cap-Clip Acid Pyrophosphatase (CELLSCRIPT, C-CC15011H) according to the induction to hydrolyze the pyrophosphate bonds of the 5’cap structures. The RNA was purified by RNA Clean & Concentrator Kits and then ligated with the 3’/5’ adapters. Adapters-ligated RNA was reversed-transcribed by using SSIV and nested RT-PCR strategy for library construction.
9
RNA-seq Analysis of CNE1 Cells
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Total RNA (1 μg) was isolated from CNE1 cells and treated with VAHTS mRNA Capture Beads (Vazyme, Nanjing, China) to enrich polyA+ RNA before constructing the RNA libraries. RNA library preparation was performed by using VAHTS mRNA-seq v2 Library Prep Kit from Illumina (Vazyme, Nanjing, China). Paired-end sequencing was performed with Illumina HiSeq 3000 at RiboBio Co., Ltd. (Guangzhou, China). For computational analysis of RNA-seq data, sequencing reads were aligned using the spliced read aligner HISAT2, which was supplied with the Ensemble human genome assembly (Genome Reference Consortium GRCh38) as the reference genome. Gene expression levels were calculated by the fragments per kilobase of transcript per million mapped reads (FPKM). Gene Set Enrichment Analysis (GSEA) was used for gene functional annotation.
10
RNA-seq Library Preparation and Sequencing
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RQ1 DNase (Promega, Madison, WI, USA) was used to remove DNA and extract total RNA. We then analyzed the purified RNA using BioRad’s Smartspec Plus (A260/A280; BioRad, Hercules, CA, USA) to measure its quality and quantity. We used integrated RNA for following experiment by 1.5% agarose gel electrophoresis method. RNA was captured using VAHTS mRNA capture beads (N401; Vazyme, Nanjing, Jiangsu, China). The purified RNAs were then used to prepare RNA-seq libraries using KAPA Stranded Kits for Illumina® platforms. We processed the cDNA products at −80 °C after size selection (300–500 bps). The cDNA libraries were then used for high-throughput sequencing (RNA-seq) using Illumina Novaseq 6000 system, and finally 150 nt paired-end sequencing reads were obtained.
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