Total RNA isolated from HCC tissues using TRIzol reagent (Invitrogen, CA, USA) was assessed using Agilent 2100 Bioanalyzer pico-RNA chips (Agilent, CA, USA). During circRNA sequencing, DNase I (Epicenter) was used to remove DNA contamination. Ribosomal RNA was removed using an Epicenter Ribo-zero rRNA Removal Kit (Epicenter, USA), and RNase R digestion (Epicenter, USA) was subsequently performed to remove linear RNA. circRNA libraries were constructed with the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations. The Illumina sequencing data were used to analyze the expression of circRNAs in tumors and adjacent tissues, using the limma package. Significantly differentially expressed circRNAs were defined as adjusted P < 0.05, and fold-change ≥ 2 or ≤0.5. A heatmap of the differentially expressed genes was generated using Cluster 3.0 software.
Agilent 2100 bioanalyzer rna pico chip
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 2100 Bioanalyzer RNA Pico chip is a lab equipment product from Agilent Technologies. It is designed for the analysis of small amounts of RNA samples. The chip allows for the separation, identification, and quantification of RNA molecules based on their size and concentration.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Mirvana microrna isolation kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rnase r digestion, by Illumina (1 mentions)
Epicenter ribo zero rrna removal kit, by Illumina (1 mentions)
Dnase 1, by Illumina (1 mentions)
Vahts total rna seq h m r library prep kit, by Illumina (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Ribominus eukaryote kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Picopure rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Smart seq v4 ultra low input rna kit for sequencing, by Takara Bio (1 mentions)
Cd45ra fitc, by BD (1 mentions)
Nucleospin rna plus xs kit, by Macherey-Nagel (1 mentions)
Facsaria fusion, by BD (1 mentions)
Complete seramir exosome rna amplification kit, by System Biosciences (1 mentions)
Experion automated electrophoresis system, by Bio-Rad (1 mentions)
Advanced analytical fragment analyzer, by Agilent Technologies (1 mentions)
Truseq stranded mrna ht kit, by Illumina (1 mentions)
10 protocols using agilent 2100 bioanalyzer rna pico chip
1
Differential circRNA Expression in HCC
2
Illumina RNA-seq Library Preparation and Sequencing
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RNA integrity and size distributions were assessed using Agilent 2100 Bioanalyzer pico-RNA chips (Agilent, CA, USA). RNA libraries were prepared according to the instructions for the VAHTS Total RNA-seq (H/M/R) Library Prep Kit from Illumina® (VAZYME, Nanjing, China). RNA-seq was performed as previously described [12 (link)], and the libraries were created and sequenced using the Illumina HiSeq 2500 platform. The RNA-seq data were uploaded to NCBI database (https://trace.ncbi.nlm.nih.gov/Traces/sra/?study =SRP165940). The accession number was NCBI: SRP165940.
3
RNA Extraction from U2OS Cells
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U2OS and U2OS-R cells were seeded at a density of 5×105 cells/well in 6-well plates and cultured for 24 h. After 24 h, cells were gently washed with serum-free DMEM. Total RNA was extracted from U2OS and U2OS-R cells using TRIzol reagent (Thermo Fisher Scientific) following a standard protocol. The integrity and size distribution of the total RNA were analyzed using an Agilent 2100 Bioanalyzer pico-RNA chip (Aglient, CA, USA).
4
RNA-seq Library Preparation from Granulosa Cells
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Total RNA from GCs was isolated using the TRIzol reagent following a standard protocol. The quality of the RNA obtained, including the integrity and size distribution, was confirmed using the Agilent 2100 Bioanalyzer pico-RNA chip (Aglient, CA, USA). Before the construction of an RNA-seq library, rRNA was removed from the total RNA samples using the RiboMinus Eukaryote Kit (Qiagen, Hilden, Germen). The NEB Next Ultra Directional RNA Library Prep Kit for Illumina (NEB, MA, USA) was used according to the manufacturer’s instructions to create an RNA library for RNA-seq. The resulting RNA-seq library was quantified using an Agilent 2100 Bioanalyzer and was run on the HiSeq 2000 platform (Illumina, CA, USA) for RNA sequencing.
5
Nucelli RNA Extraction and Amplification
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RNA extraction from LAM samples including DNA digest was done using PicoPure RNA isolation kits (ThermoFisher Scientific) following the manufacturer’s instructions, except that 15 µl extraction buffer was used. Each sample contained pooled individuals from the same accession to obtain 38–226 nucelli sections per sample (Supplementary Table S1 at JXB online). The RNA integrity of controls was determined using Agilent 2100 Bioanalyzer RNA Pico Chips. Good and reproducible RNA integrity was consistently achieved (Supplementary Fig. S1A ).
A SMARTseq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio USA) was used for linear amplification of mRNA derived from nucelli samples following the manufacturer’s instructions (Supplementary Fig. S1B, C ). Amplified cDNA was purified using AMPure Sample Purification Beads (Beckman Coulter, Brea, USA) and eluted in nuclease-free H2O and stored at –20 °C until subsequent library preparation.
A SMARTseq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio USA) was used for linear amplification of mRNA derived from nucelli samples following the manufacturer’s instructions (
6
Fusobacterium nucleatum Invasion Assay
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To further validate gene expression during Fusobacterium F. nucleatum F. nucleatum F. nucleatum
7
Profiling Cerebrospinal Fluid and Blood CD4+ T Cells
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CD4+ T cells from paired CSF and blood samples were stained using these following antihuman antibodies: CD4-APC, CD45RA-FITC (BD Biosciences), and CXCR5-PeVio615 (Miltenyi Biotec). cTfh cells and memory non-Tfh cells were defined as CD4+CD45RA−CXCR5+ and CD4+CD45RA−CXCR5− T cells, respectively. Both groups of cells were sorted using an FACSAria Fusion (BD Biosciences). At least 2,000 cells were necessary to obtain sufficient amount of RNA. RNA was isolated from sorted cTfh cells and memory non-Tfh cells using NucleoSpin RNA Plus XS kit (Macherey-Nagel, Düren, Germany). Total RNA concentration and integrity were assessed using the Agilent 2100 Bioanalyzer RNA Pico chip (Agilent Technologies, Santa Clara, CA).
8
Extraction and Amplification of Adipocyte-Exosomal RNA
9
Isolation and Analysis of Adipocyte-Derived Exosomal miRNA
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Example 2
Isolation of microRNA from the Adipocyte-Derived Exosomes Obtained from Urine and Blood/Serum. Total RNA from the adipocyte-derived exosomes purified above was then isolated using the mirVana™ microRNA Isolation Kit (Life technologies, Frederick, Md.), in accordance with the manufacturer's directions. For some experiments, the extracted RNA was then amplified using the Complete Seramir® Exosome RNA Amplification Kit from Media and Urine (System Biosciences, Mountain View, Calif.) according to the manufacturer's directions, and RNA quality was measured using an Agilent® 2100 Bioanalyzer RNA Pico Chip (Agilent Technologies, Santa Clara, Calif.). Information on the type and abundance of RNAs in the adipocyte-derived exosomes was then determined using microarray analysis.
10
RNA Extraction and Sequencing from PAXgene Samples
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Prior to RNA extraction, frozen whole-blood PAXgene samples were thawed at room temperature for 2 h and subjected to RNA extraction using PAXgene Blood miRNA Kit (PreAnalytix/QIAGEN, Cat# 763134). Total RNA, including RNA longer than approximately 18 nucleotides, was purified, following the protocol supplied by the kit manufacturer. Sample concentration was measured with Nanodrop 2000 spectrophotometer and Qubit Fluorometric Quantitation (Thermo Fisher Scientific). The quality of the samples was ensured with Experion Automated Electrophoresis System (Bio Rad) and Agilent 2100 Bioanalyzer RNA Pico chip. Library preparation and sequencing were carried out at the Finnish Functional Genomics Centre (FFGC). Before starting library preparation, ERCC Spike-in Mix 1 (Invitrogen P/N 4456739) was added to 100 ng RNA according to the kit's protocol. RNA-seq libraries were prepared using TruSeq stranded mRNA HT kit and protocol # 15031047 (Illumina). The quality and quantity of the amplified libraries were measured using Advanced Analytical Fragment Analyzer (Agilent) and Qubit Fluorometric Quantitation, respectively. Pooled libraries were sequenced on an Illumina NovaSeq 6000 instrument, using 2 × 50 bp paired-end sequencing.
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