Five out of 47 pairs of specimens were selected for sequencing. The preparation of whole transcriptome libraries and deep sequencing were conducted by Hua Da Gene Corp., Ltd. (Shenzhen, China). The whole transcriptome libraries were established using the New England Biolabs (NEB) Next Ultra Directional RNA Library Prep Kit for Illumina according to the manufacturer’s instructions. Libraries were tested for quality using the Agilent 2100 Bioanalyzer system and the StepOnePlus RT-PCR (Applied Biosystems, USA) system. The qualified libraries were sequenced on an Illumina HiSeq X Ten instrument in paired-end form, generating 150 nucleotides. Clean reads were aligned to the human genome (GRCh38) using the HISAT program (18 (link)) and alignment results were reconstructed with cuff compare. The NONCODE and Pfam databases were used as annotation references for lncRNA and mRNA analyses. INFERNAL (19 (link)) was used to predict lncRNAs. CIRI (20 (link)) and find_circ (10 (link)) were used to predict circRNAs. The read count of each transcript was normalized in the form of fragments per kilobase of exon per million mapped reads (FPKM). In our study, differentially expressed genes and transcripts were identified using the following criteria: |log2(fold-change)| ≥1 and P value ≤0.05 between the two samples.
Next ultra directional rna library prep kit
Manufactured by Illumina
Sourced in United States
The Next Ultra Directional RNA Library Prep Kit is a laboratory instrument designed for the preparation of DNA libraries from RNA samples. The kit provides the necessary reagents and protocols for the conversion of RNA into cDNA and the subsequent generation of sequencing-ready libraries.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Steponeplus rt pcr, by Thermo Fisher Scientific (1 mentions)
Hiseq x ten, by Illumina (1 mentions)
Hiseq pe cluster kit, by Illumina (1 mentions)
Epicenter ribo zero rrna removal kit, by Illumina (1 mentions)
Rnase r, by Illumina (1 mentions)
Hiseq 4000 platform, by Illumina (1 mentions)
Epicentre ribo zero rrna removal kit, by Illumina (1 mentions)
Qubit rna assay kit, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Magna rip rbp immunoprecipitation kit, by Merck Group (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Ribominus eukaryote kit, by Qiagen (1 mentions)
Rq1 dnase, by Promega (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nanophotometer, by Implen (1 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (1 mentions)
Ribo zero gold kit, by Illumina (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
7 protocols using next ultra directional rna library prep kit
1
Transcriptome Profiling of Biological Specimens
2
Ribo-zero and RNase R RNA-seq
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A total of 5 μg RNA was treated with Epicenter Ribo-zero rRNA Removal Kit and RNase R (Epicenter). Sequencing libraries were generated using the Next Ultra Directional RNA LibraryPrep Kit for Illumina (NEB), following the manufacturer’s recommendations. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using HiSeq PE cluster kit (Illumina). After cluster generation, the library preparations were sequenced on an Illumina HiSeq 4000 platform, and 150-bp paired-end reads were generated.
3
Transcriptome Profiling via RNA-seq
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The quality of all participants' RNA (including participants who are sequenced first and participants who are verified later) were evaluated by a nanometer photometer spectrophotometer (IMPLEN). The Qubit RNA Assay Kit with the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Inc.) and an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.) were used to determine the RNA concentration and examine the RNA integrity, respectively. Subsequently, the RNA library was constructed using a total amount of 3 µg of RNA each sample, and its RNA integrity number was >7.0. Ribosomal RNA (rRNA) was removed by the Epicentre Ribo-zero rRNA Removal kit (Epicentre; Illumina, Inc.) according to the manufacturer's protocol. Afterwards, the strand-specific sequencing libraries was obtained by the New England Biolabs (NEB) Next Ultra Directional RNA Library Prep Kit for Illumina (NEB) using the dUTP method. The RNA-seq assay was accomplished on an Illumina Hiseq 2000 platform (Illumina, Inc.) and 100 bp paired-end reads were obtained. The preparation of the total transcriptome libraries was completed by YuXi Bioinformatics Corp.
4
RNA Immunoprecipitation and Sequencing
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For RIP, control and FXR1-knockdown T24 cells were harvested and lysed in RIP lysis buffer (Magna RIP RBP Immunoprecipitation Kit, Millipore). After centrifugation, the supernatant was incubated with antibody-conjugated and agarose beads (Roche, USA) overnight at 4 °C. The bead-bound RNAs were extracted with TRIzol and subjected to RIP-seq or qRT-PCR analysis using the primer pairs indicated in Supplementary Table S4 . The libraries of immunoprecipitated RNA and input RNA were made using the NEB Next Ultra Directional RNA Library Prep Kit and sequenced on the Illumina HiSeq platform (Illumina, California, USA).
5
RNA Sequencing of U2OS Cell Lines
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Total RNA samples obtained from U2OS and U2OS-R cells were treated with RQ1 DNase (Promega, USA) to minimize DNA contamination. Next, rRNA was removed from the total RNA samples using the RiboMinus Eukaryote Kit (Qiagen, Hilden, Germen) in accordance with the manufacture’s instruction. Two libraries were created using Next Ultra Directional RNA Library Prep Kit for Illumina (NEB, MA, USA) as per the manufacturer’s instructions. To obtain circRNA reads, libraries were subjected to HiSeq 2000 platform for RNA-seq after quantifying the libraries using an Agilent 2100 Bioanalyzer (Agilent, CA, USA).
6
High-Throughput RNA-Seq Protocol for Transcriptome Analysis
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The high-throughput RNA-Seq experiments were performed by Annoroad Gene Technology (Beijing, China). Briefly, total RNAs was extracted from cells by TRIzol Reagent (Life Technologies). Then the RNA samples were evaluated for degradation and impurities by 1% agarose electrophoresis. The RNA purity was determined using a NanoPhotometer (IMPLEN, CA, USA). An Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano Kit were used to assess RNA concentration and integrity (Agilent Technologies, CA, United States). The samples were sequenced and samples with RNA Integrity Number (RIN) scores > 9.5 were used in subsequent analyses. The mouse genome was selected as the reference genome in this study. High -throughput sequencing was performed by Annoroad technologies (Beijing, China). The lncRNA library was constructed from total RNA (2 μg) using different index tags with the next Ultra directional RNA library prep Kit for Illumina (NEB, Ipswich, USA) according to the manufacturer's instructions. Data quality control involved removal of contaminated reads, low quality reads, reads containing N ratios >5%, and reads that matched the ribosomal RNA. The RNA-seq data filtered for each sample was compared with the genome by HiSAT2. FPKM was used to estimate gene expression levels quantitatively.
7
RNA-seq Analysis of Chk1 Knockdown in Breast Cancer Cells
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Three pairs of MDA-MB-231 cells in si-CHK1 and si-control groups were prepared for RNA-seq (Beijing Annoroad Co. Ltd). Total RNA was extracted with TRIzol® (Invitrogen, Carlsbad, CA, USA). We depleted ribosomal RNA (rRNA) from RNA samples in using Ribo-Zero Gold Kits (Illumina, US). In accordance with the protocol of New England Biolabs (NEB; Ispwich, Massachusetts, US) Next Ultra Directional RNA Library Prep Kit for Illumina, RNA libraries were established and sequenced as 150 bp paired-end reads using the HiSeq X ten. As described elsewhere 8 (link), we filtered RNA-seq next-generation sequencing (NGS) reads to obtain clean reads for further evaluation and analysis, including quality inspection of reads according to Phred Score, comparison to the human genome reference assembly (hg19) using HiSAT2 and merger of transcripts in StringTie. We used fragments per kilobase of transcript per million mapped reads (FPKM) to assess mRNA expression. Finally, the heatmap was generated using R software with differentially expressed genes (fold change > 2 or < 0.5, P < 0.1).
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