Total RNA was extracted from TMZ-sensitive/resistant U87MG cell lines by TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. RNA quality was assessed by Nanodrop2000 and Qubit 3.0. RNA integrity was determined by Agilent 2100 Bioanalyzer. mRNA Capture Beads (Vazyme Biotech, Nanjing, China) were used to eliminate rRNAs and a VAHTS Total RNA-Seq Library Preparation Kit (Vazyme Biotech) was used to prepare libraries. Sequencing was performed on the Illumina Hiseq 2500 platform (pair-end 150 bp). The raw sequencing data have been deposited in the NCBI (BioProject accession: PRJNA768121). Data were further processed by R (version 4.1.0). A heatmap of gene expression profiles was generated using the pheatmap package. Principal component analysis (PCA) of each sample was performed and the top two principal components were shown. Differentially expressed genes (DEGs) were identified using the limma package (| log2(fold change) | > 1 and adjusted p-value < 0.05 as threshold). A volcano plot was illustrated to visualize the distribution of DDR genes using the EnhancedVolcano package.
Mrna capture beads
Manufactured by Vazyme
Sourced in China
MRNA Capture Beads are magnetic beads designed for the efficient capture and purification of mRNA from biological samples. The beads have a high affinity for the polyA tails of mRNA molecules, allowing for selective isolation and enrichment of mRNA from total RNA extracts.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Vahts total rna seq library preparation kit, by Vazyme (2 mentions)
Dna clean beads, by Vazyme (2 mentions)
Hiseq 2500, by Illumina (1 mentions)
Nanophotometer spectrophotometer, by Implen (1 mentions)
Hiseq 3000, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Rna extraction kit, by Tiangen Biotech (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
Nanodrop 2000, by Agilent Technologies (1 mentions)
Hipure yeast rna kit, by Magen Biotechnology Co (1 mentions)
Qubit 3.0 fluorescence quantifier, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by New England Biolabs (1 mentions)
Hiscribe t7 arca mrna kit, by New England Biolabs (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Trizol solution, by Tiangen Biotech (1 mentions)
Vahts universal v6 rna seq library prep kit, by Vazyme (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Low melting point agarose, by Thermo Fisher Scientific (1 mentions)
10 protocols using mrna capture beads
1
Transcriptomic Analysis of TMZ-Sensitive/Resistant Glioblastoma
2
Transcriptome Profiling by Single-Molecule Sequencing
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Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, United States). RNA degradation was detected using 1% agarose gel electrophoresis, while RNA purity and concentration were determined using NanoPhotometer® spectrophotometer (Implen, München, Germany). mRNA was then captured and purified from total RNA with mRNA Capture Beads (Vazyme Biotech, Nanjing, China). After fragmenting the mRNA into 100∼200 nt in the Fragmentation Buffer, mRNA was reverse-transcribed into cDNA. Synthesized cDNA was purified using DNA Clean Beads (Vazyme Biotech).
We used a sequencing strategy with optimized single-molecule barcodes to minimize sequence dependent bias and amplification noise (Shiroguchi et al., 2012 (link); Ogawa et al., 2017 (link)). The Purified cDNA was ligated to the adaptor with UMI (Unique molecular identifier) and purified using DNA Clean Beads. Finally, cDNA libraries were constructed from cDNA using PCR amplification (NEBNext Ultra RNA Library Prep Kit, Ipswich, MA, United States). The qualities of cDNA libraries were detected by the Agilent Bioanalyzer 2100 (DNA Technologies Core, Davis, CA, United States).
We used a sequencing strategy with optimized single-molecule barcodes to minimize sequence dependent bias and amplification noise (Shiroguchi et al., 2012 (link); Ogawa et al., 2017 (link)). The Purified cDNA was ligated to the adaptor with UMI (Unique molecular identifier) and purified using DNA Clean Beads. Finally, cDNA libraries were constructed from cDNA using PCR amplification (NEBNext Ultra RNA Library Prep Kit, Ipswich, MA, United States). The qualities of cDNA libraries were detected by the Agilent Bioanalyzer 2100 (DNA Technologies Core, Davis, CA, United States).
3
RNA Sequencing of Breast Cancer Cells
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When extracted from the breast cancer cells, total RNA was treated with mRNA Capture Beads (Vazyme) so that polyA RNA was enriched. Based on the VAHTS mRNA‐seq v2 Library Prep Kit for Illumina (Equitech‐Bio), an RNA library was prepared, and the paired‐end sequencing was done by RiboBio Co., Ltd. with Illumina HiSeq 3000. To perform the analysis of the RNA‐sequencing data, sequencing reads were compared on the spliced‐reads comparator of HISAT2, based on the human genome collection as a reference genome. The gene expression level of each transcript was set as reads per million exon model per kilobase. Gene Set Enrichment Analysis (GSEA) was applied to the annotation of gene function. When the gene expression multiple changes were >2 with a p value <0.05 through Cufflinks calculation, the genes were considered to be expressed differentially and significantly.
4
Transcriptomic Profiling of Temozolomide-Resistant Glioblastoma
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Total RNA was extracted from temozolomide (TMZ) sensitive/resistant U87MG cell lines using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). RNA quality was assessed using Nanodrop2000 and Qubit 3.0. RNA integrity was determined by Agilent 2100 Bioanalyzer. Then, total RNA was treated with mRNA Capture Beads (Vazyme Biotech, Nanjing, China) to eliminate rRNAs. A VAHTS Total RNA-Seq Library Preparation Kit (Vazyme Biotech) was used for library preparation. RNA sequencing was performed on the Illumina Hiseq 2500 platform. Pair-end reads were generated with reading lengths up to 150 bp. Differentially expressed genes were identified by limma package in R (logFC > 1 and P < 0.05 as threshold). The heatmap was illustrated to visualize the levels of differential expressed DNA replication-related genes. The volcano plot showed the differential distribution of DNA replication and repair-related genes. The raw sequencing data have been deposited in the NCBI under BioProject accession number PRJNA768121.
5
RNA Extraction and Sequencing Workflow
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The total RNA was extracted by using RNA Extraction Kit (Tiangen, Beijing, China). The quality of RNA was detected by RNase-free agarose gel electrophoresis. The concentration and purity of the total RNA samples were detected by NanoDrop 2000, followed by calculation of 28S/18S ratio and RIN value through Agilent 2100 (Agilent Technologies, Santa Clara, Technologies, CA, United States). Poly (A) mRNA was purified and enriched from total RNA by mRNA capture beads (kits bought from Vazyme, Nanjing, China). Reverse transcription primers were used to bind to the poly-A tail of mRNA by annealing. Double cDNA strand was amplified by PCR amplification primers. The DNA damage repair and plus-A reaction were done by using NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair/dA-Tailing Module, respectively. Connections of sequencing adaptors were done by using the SQK-LSK109 kit (ONT). A total of 18 RNA-Seq libraries were constructed and sequenced.
6
Yeast RNA extraction and mRNA sequencing
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Yeast cells were grown overnight in YPD or SD medium at 30 °C, diluted to an OD600 of 0.2 and collected when the OD600 reached 0.8. Cells were harvested by spinning down at 3000 r.p.m. for 5 min, and then, the cell pellets were washed with 10 mL sterile water twice. Total RNA was extracted with HiPure Yeast RNA Kit (Magen, Guangzhou, China, R4182‐02). The purity and yield of RNAs were examined by NanoDrop One Spectrophotometer (Gene Company). RNA integrity was examined by agarose electrophoresis.
For preparation of mRNA, 20 μg of total RNA was diluted into 50 μL of DEPC water and incubated with 50 μL of mRNA capture beads as described in the product manual (Vazyme, Nanjing, China, N401‐01). Then, the enriched mRNA was used for strand‐specific library construction by the KAPA Stranded RNA Sequencing (RNA‐Seq) Library Preparation Kit (KAPA, KK8401) through the dUTP method. The libraries were used to generate a total of ˜ 6 GB sequencing data from 75‐bp length single‐end reads using a NextSeq CN500 equipment.
For preparation of mRNA, 20 μg of total RNA was diluted into 50 μL of DEPC water and incubated with 50 μL of mRNA capture beads as described in the product manual (Vazyme, Nanjing, China, N401‐01). Then, the enriched mRNA was used for strand‐specific library construction by the KAPA Stranded RNA Sequencing (RNA‐Seq) Library Preparation Kit (KAPA, KK8401) through the dUTP method. The libraries were used to generate a total of ˜ 6 GB sequencing data from 75‐bp length single‐end reads using a NextSeq CN500 equipment.
7
RNA-Seq Library Preparation Protocol
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The 3 μg of RNA per sample was used as the input material for RNA sample preparation. mRNA was separated from total RNA and purified by mRNA Capture Beads (Vazyme, Nanjing, China). The purified mRNA was incubated in a preheated Polymerase chain reaction (PCR) instrument (Life Technologies, CA, USA) at 94°C for 7 min, interrupted, cooled immediately after the interruption, and centrifuged. The first strand of cDNA was synthesized using the supernatant containing fragment RNA as the template. The double‐strand synthesis reaction reagents were added to the first strand product of the synthesized cDNA, mixed, centrifuged, and incubated in a metal bath to synthesize the second strand. Magnetic beads were used to purify the double‐strand cDNA product. After adapter ligation, the fragments were screened by DNA clean beads (Vazyme, Nanjing, China), and the library was enriched by PCR using the screened cDNA fragments as templates.
Qubit 3.0 fluorescence quantifier (Thermo Fisher Scientific, Waltham, MA, USA) was used for preliminary quantification until the concentration reached more than 1 ng/μL. The Qsep400 high throughput analysis system was used to detect the inserted fragments of the library. After the pieces were inserted as expected, Q‐PCR was used to accurately quantify the effective concentration of the library (effective concentration>2 nM).
Qubit 3.0 fluorescence quantifier (Thermo Fisher Scientific, Waltham, MA, USA) was used for preliminary quantification until the concentration reached more than 1 ng/μL. The Qsep400 high throughput analysis system was used to detect the inserted fragments of the library. After the pieces were inserted as expected, Q‐PCR was used to accurately quantify the effective concentration of the library (effective concentration>2 nM).
8
Isolation and Purification of mRNA
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Total RNA was extracted from HEK293T cells with TRIzol (Invitrogen, 15,596,026), according to the manufacturer’s instructions. The resulting total RNA was treated with DNase I (NEB, M0303) to avoid genomic DNA contamination. Phenol/chloroform extraction and ethanol precipitation were then performed to purify and concentrate total RNA. For mRNA isolation, mRNA Capture Beads (Vazyme, N401-01) were used according to the manufacturer’s instructions. Cre mRNA was prepared by in vitro transcription of synthetic Cre DNA with HiScribe T7 ARCA mRNA Kit (NEB, E2060S) according to the manufacturer’s instructions, then treated with DNase I (NEB) to digest DNA template.
9
RNA-seq Analysis of ADSCs
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ADSCs were harvested, and total RNA was extracted using TRIzol solution (TIANGEN, China). The concentration of RNA was measured using Nanodrop 2000 and evaluated using Agilent 2100 (LabChip GX). mRNA was purified using mRNA Capture Beads (Vazyme, China), reverse transcribed into cDNA using the Script RT reagent kit (Vazyme, China), and then synthesized into dsDNA. The dsDNA library was constructed using VAHTS Universal V6 RNA-seq Library Prep Kit (Vazyme) and purified using VIHTS DNA Clean Beads (Vazyme) according to the manufacturer’s instructions. The qualified dsDNA samples were sequenced using Illumina Nova Seq 6000 platform (San Diego, USA). Raw sequencing reads were mapped to GRCh38 assembly of the human genome using Tophat2, version 2.0.10 [33 (link)]. Fragments per kilobase of exon model per million mapped reads (FPKM) were calculated using Stringtie and normalized with TMM [34 (link)–36 (link)]. Differentially expressed genes (DEGs) were analyzed using the DESeq 2 package, version 1.10.1 [37 (link)]. Genes with log-fold change > 1.5 and false discovery rate (FDR) < 0.05 were considered significant transcriptomic changes. This sequencing dataset is available at GEO: GSE222263.
10
Comprehensive RNA Isolation and Library Preparation
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Yeast-tRNA (Roche 10109525001), RNase inhibitor (Promega N251A), RNaseZap (Invitrogen AM9780), TRIzol Reagent (Invitrogen 15596018), 3 M Sodium Acetate pH5.5 (Invitrogen AM9740), Perfect Start Green qPCR SuperMix (TransGene AQ601-04), RNeasy RNA purification Kit (Qiagen 74104), mRNA Capture Beads (Vazyme N401), Qubit RNA Assay Kit (Qubit Q32852), RNA secure Reagent (Ambion AM7006), Turbo DNase (Invitrogen AM2238), riboPOOLs (rRNA removal probes) (siTOOLs Biotech), Streptavidin magpoly beads (SMART lifesciences SM01710), Low Melting Point Agarose (Invitrogen 16520-050), 10 × TBE Buffer (Invitrogen 15581-044), DNA size Marker (DL1000, Takara 3591Q), NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB E7760).
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