Agilent 2100 bioanalyzer

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The Agilent 2100 Bioanalyzer is a lab instrument that provides automated analysis of DNA, RNA, and protein samples. It uses microfluidic technology to separate and detect these biomolecules with high sensitivity and resolution.

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15 883 protocols using agilent 2100 bioanalyzer

Sequence Libraries Preparation from Total RNA

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Total RNA was extracted with the TRI reagent (Ambion) using the manufacturer’s instructions. The RNase inhibitor RiboLock (Fermentas) was added to the samples and DNA was removed by treatment with DNase I (Fermentas). The integrity of the RNA samples was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies). Subsequently, the 23S, 16S, and 5S rRNAs were removed by subtractive hybridization using the MICROBExpress kit (Ambion) following the protocol reported by Gómez-Lozano et al. (2014) (link). Removal of the rRNA was confirmed by an analysis with an Agilent 2100 Bioanalyzer (Agilent Technologies). Sequencing libraries were prepared using the TruSeq Stranded mRNA Sample Preparation kit (Illumina). After each step, the samples were validated using an Agilent 2100 Bioanalyzer (Agilent Technologies) and the final RNA concentration was measured using a Qubit 2.0 Fluorometer (Invitrogen). The libraries were sequenced using the Illumina HiSeq2000 platform with a paired-end protocol and read lengths of 100 nucleotides.

Rubio-Gómez J.M., Santiago C.M., Udaondo Z., Garitaonaindia M.T., Krell T., Ramos J.L, & Daddaoua A. (2020). Full Transcriptomic Response of Pseudomonas aeruginosa to an Inulin-Derived Fructooligosaccharide. Frontiers in Microbiology, 11, 202.

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RNA Extraction and Sequencing Protocol

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Total RNA was extracted from samples before sequencing using TRIzol reagent (Invitrogen, San Diego, CA, USA). RNA integrity and quantity were evaluated by using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California, USA). The cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and random primers, with enriched and fragmented RNA as template. The cDNA was converted to dsDNA for library construction. The libraries were quantified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California, USA). Sequencing was performed using Illumina NextSeq6000 platform (Illumina, San Diego, CA, USA). Multiplex Small RNA Library Prep Set for Illumina was used to construct small RNA libraries based on miRNA sequencing, in accordance with the manufacturer’s protocol. Fragments (18-35 nt) were extracted from the total RNA, and indices and adaptors were added for PCR amplification. Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) was used for library quantification. The library was sequenced by Shanghai Personal Biotechnology Co. Ltd. (Shanghai, China) using Hiseq platform (Illumina).

Yang B., Huang X., Xu S., Li L., Wu W., Dai Y., Ge M.X., Yuan L., Cao W., Yang M., Wu Y, & Deng D. (2021). Decreased miR-4512 Levels in Monocytes and Macrophages of Individuals With Systemic Lupus Erythematosus Contribute to Innate Immune Activation and Neutrsophil NETosis by Targeting TLR4 and CXCL2. Frontiers in Immunology, 12, 756825.

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DNA Library Preparation and Size Selection

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We constructed a library containing 300–390 ng DNA for each sample. Libraries with barcodes were size-selected from within the range of 190–240 bp (insert DNA ranging from 100 to 150 bp) using E-Gel CloneWell Agarose Gels (Invitrogen, Carlsbad, CA, USA). A piece of E-Gels contains six effective wells, each well can run a mixed sample which contains five samples of the NIPS DNA sequencing library. So, a piece of E-Gels can run thirty samples. Per the manufacturer’s instructions: 100 ng DNA per well was loaded on E-Gel EX Gels, 2% (Invitrogen, Carlsbad, CA, USA), a pre-cast 2% agarose gel with 0.8% SYBR stain; the gel was run on E-Gel iBase Power System (Invitrogen, Carlsbad, CA, USA) for approximately 15 min; and DNA with target sizes was retrieved from the bottom wells on the gel. A 50 bp DNA ladder was used as the marker (Invitrogen). The selected library was then tested with an Agilent 2100 Bioanalyzer (Fig. 5) and quantified by real-time polymerase chain reaction (PCR) using KAPA Library Quantification Kits (for the Ion Torrent platform).Figure 5

Selection of the library using E-gels and testing by Agilent 2100 Bioanalyzer. (A) Selection of the library between 190 and 240 bp (insert DNA was from 100 to 150 bp). (B) The selected library was tested using an Agilent 2100 Bioanalyzer.

Liang B., Li H., He Q., Li H., Kong L., Xuan L., Xia Y., Shen J., Mao Y., Li Y., Wang T, & Zhao Y.L. (2018). Enrichment of the fetal fraction in non-invasive prenatal screening reduces maternal background interference. Scientific Reports, 8, 17675.

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RNA and miRNA Quality Assessment

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RNA concentration and purity were measured by Nanodrop ND 1000 spectrophotometer (Thermo Scientific, Waltham, MA 02451, USA) using 1 µL of isolated RNA. The A280/260 and A260/230 absorbance ratios were used to assess purity, considering a ratio between 1.8 and 2.0 to be pure.
RNA and miRNA integrity was estimated by microcapillary electrophoresis in an Agilent 2100 Bioanalyzer (Agilent Technology, Santa Clara, CA 95051, USA). For miRNA analysis, sample aliquots were diluted at 10 ng/µL just before use and measured in the Agilent 2100 Bioanalyzer using a Small RNA kit (Agilent Technology, Santa Clara, CA 95051, USA). The integrity of miRNA was calculated as the relative abundance of miRNA species (10–40 nt) in comparison to the total amount of small RNA fraction (10–150 nt). For RNA integrity, 1 µL of RNA was submitted to Agilent 2100 Bioanalyzer using Agilent RNA 6000 nano kit (Agilent Technology, Santa Clara, CA 95051, USA). RIN number and relative percentage of total RNA species (60–149 nt and 150–299 nt) were recorded.

Azzalini E., De Martino E., Fattorini P., Canzonieri V., Stanta G, & Bonin S. (2019). Reliability of miRNA Analysis from Fixed and Paraffin-Embedded Tissues. International Journal of Molecular Sciences, 20(19), 4819.

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RNA-seq Library Preparation for Illumina Sequencing

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RNA extraction was performed with the Qiagen RNEasy Miniprep kit according to the manufacturer protocol (Qiagen, Cat. 74134). RNA quality was determined using an Agilent 2100 Bioanalyzer and all samples were found to have RNA integrity numbers (RIN) ≥ 9.2. Sequencing libraries were prepared as described previously (59 (link)) by the NC State Genome Science Lab (GSL), using NEBNext Ultra Directional RNA Library Prep kit and NEBNext Poly(A) mRNA Magnetic Isolation Module (catalogs E7420 and E7490; New England Biolabs, Ipswich, MA, USA) for Illumina sequencing. Isolation, heat fragmentation and priming were performed according to manufacturer instructions. cDNA synthesis was followed by purification and size selection. Finally, library clean-up was performed used AMPure XP beads (Beckman Coulter Genomics, Brea, CA, USA; Cat. A63881) and quality was assessed using the Agilent 2100 Bioanalyzer. For each sample, library sequencing was performed using a 50-bp paired-end protocol on a single lane of an NovaSeq6000 sequencer. Approximately, 50 to 90 million uniquely mapped reads were generated per fetal forebrain library. The raw data were deposited in NCBI’s Gene Expression Omnibus (the ID will be placed here upon manuscript acceptance).

Witchey S.K., Doyle M.G., Fredenburg J.D., St. Armour G., Horman B., Odenkirk M.T., Aylor D.L., Baker E.S, & Patisaul H.B. (2022). Impacts of Gestational FireMaster 550 (FM 550) Exposure on the Neonatal Cortex are Sex Specific and Largely Attributable to the Organophosphate Esters. Neuroendocrinology, 113(12), 1262-1282.

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Transcriptome Profiling of Bovine PBMCs

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Total RNA was then isolated from PWBCs of 23 heifers using TRIzol™ reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. RNA yield was quantified using the Qubit™ RNA Broad Range Assay Kit (Eurogene, OR) on a Qubit® Fluorometer, and integrity was assessed on Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA) using an Agilent RNA 6000 Nano kit (Agilent, Santa Clara, CA). We obtained RIN values ranging between 7.7 and 8.8. Furthermore, samples with rRNA ratios (28S:18S) greater than 1.5 were further processed for library construction (range 1.5–1.8). Libraries were prepared with the TruSeq Stranded mRNA Library Prep kit (Illumina, Inc., San Diego, CA) following manufacturer’s instructions. Libraries were quantified with Qubit™ dsDNA High Sensitivity Assay Kit (Eurogene, OR) and quality was evaluated using the High Sensitivity DNA chip (Agilent, Santa Clara, CA) on an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA). Libraries were sequenced in a HiSeq 2500 system at the Genomic Services Laboratory at HudsonAlpha, Huntsvile, AL to generate 125 nucleotide long pair-end reads.

Dickinson S.E., Griffin B.A., Elmore M.F., Kriese-Anderson L., Elmore J.B., Dyce P.W., Rodning S.P, & Biase F.H. (2018). Transcriptome profiles in peripheral white blood cells at the time of artificial insemination discriminate beef heifers with different fertility potential. BMC Genomics, 19, 129.

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Transcriptome Analysis of Honey Bee Larvae

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Total RNA of each sample was extracted using TRIzol reagent (Tiangen, Beijing). The concentration and integrity of RNA were assessed using Qubit 3.0 Fluorometer and Agilent 2100 bioanalyzer. Each group of queen and worker larvae had 3 biological replicates.
Total mRNA of each sample was enriched using oligo (dT) magnetic beads. First-strand cDNA was synthesized by random hexamers, and the second-strand cDNA was synthesized in DNA polymerase I system using dNTPs and RNaseH. Afterwards, cDNA was subjected to end repair and performed with poly(A) tail and ligation sequencing adapter. 200–350 bp cDNA were selected by AMPure XP beads, and then were PCR amplified and purified by AMPure XP beads. Library quality was evaluated using Agilent 2100 bioanalyzer and qRT-PCR. Totally 18 libraries were sequenced by an Illumina NovaSeq 6000 platform. This was performed by Wuhan Benagen Tech Solutions Company Limited also.

He X.J., Barron A.B., Yang L., Chen H., He Y.Z., Zhang L.Z., Huang Q., Wang Z.L., Wu X.B., Yan W.Y, & Zeng Z.J. (2022). Extent and complexity of RNA processing in honey bee queen and worker caste development. iScience, 25(5), 104301.

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RNA-seq Analysis of Differentiated Cells

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After 28 days of differentiation, total RNA was extracted from cells by using the RNeasy Plus Mini Kit (Qiagen). The sequencing data was provided by Shanghai Genechem Co. RNA quantity and integrity were verified by using the Agilent 2100 Bioanalyzer. The RNA-seq library was prepared with the NEBNext Ultra RNA Library Prep Kit for Illumina according to the protocol provided by the manufacturer. The library was assessed by using the Agilent 2100 Bioanalyzer. Finally, sequencing was performed on an Illumina HiSeq sequencer with a 150-bp paired-end sequencing reaction. Primary sequencing data from Illumina HiSeq sequencing were referred for raw read quality control. The clean reads obtained were used for downstream bioinformatics analysis. Then the clean reads were aligned to the reference sequences by using HISAT2 software. The expression levels of genes were determined according to the value of fragments per kilo bases per million fragments (FPKM). We then used DESeq R package to filter differentially expressed genes (DEGs). After statistical analysis, we screened DEGs as follows: |log2foldchange|>1, with p < 0.05.

Jin M., Yi X., Zhu X., Hu W., Wang S., Chen Q., Yang W., Li Y., Li S., Peng Q., Pan M., Gao Y., Xu S., Zhang Y, & Zhou S. (2024). Schisandrin B promotes hepatic differentiation from human umbilical cord mesenchymal stem cells. iScience, 27(2), 108912.

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Transcriptome Analysis of CPB Lifecycle

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Total RNA from CPB larvae, pupae and moth were extracted using the GeneAll Hybrid-R™ kit (GeneAll Biotechnology, Seoul, Korea) according to the manufacturer's instructions. RNA Integrity Number (RIN) was determined using RNA Nano 6000 Assay Kit (Agilent Technologies, CA, USA) with the Agilent 2100 Bioanalyzer (Agilent Technologies). The libraries were prepared for 150bp paired-end sequencing using TruSeq stranded mRNA Sample Preparation Kit (Illumina, CA, USA). Namely, mRNA molecules were purified and fragmented from 1μg of total RNA using oligo (dT) magnetic beads. The fragmented mRNAs were synthesized as single-stranded cDNAs through random hexamer priming. By applying this as a template for second strand synthesis, double-stranded cDNA was prepared. After sequential process of end repair, A-tailing and adapter ligation, cDNA libraries were amplified with PCR (Polymerase Chain Reaction). Quality of these cDNA libraries was evaluated with the Agilent 2100 Bioanalyzer (Agilent, CA, USA). They were quantified with the KAPA library quantification kit (Kapa Biosystems, MA, USA) according to the manufacturer's library quantification protocol. Following cluster amplification of denatured templates, sequencing was progressed as paired-end (2×150bp) using Illumina NovaSeq6000 (Illumina, CA, USA).

Tan C.L., Kasran R., Lee W.W., & Leong W.M. (2021). Transcriptome analysis of developmental stages of cocoa pod borer, Conopomorpha cramerella: A polyphagous insect pest of economic importance in Southeast Asia.

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Total RNA Extraction and RNA-seq Library Preparation

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Total RNA was extracted using mirVana^TM miRNA Isolation kit (Cat# AM1561, Ambion) following the manufacturer’s instructions. Quantity and quality of RNA samples were determined using a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, United States) and Agilent 2100 Bioanalyzer (Agilent Technologies, United States). TruSeq^®Stranded Total RNA Sample Preparation kit was used to prepare libraries following the manufacturer’s instructions, and purified libraries were quantified using a Qubit 2.0^®Fluorometer (Life Technologies, United States) and Agilent 2100 Bioanalyzer (Agilent Technologies, United States). Clusters were generated by cBot library and sequenced using an Illumina HiSeq 2500 (Illumina, United States). Sequencing was performed at Origin-Biotech Inc. (Ao-Ji Biotech, Shanghai, China).

Duan X., Han L., Peng D., Chen W., Peng C., Xiao L, & Bao Q. (2019). High Throughput mRNA Sequencing Reveals Potential Therapeutic Targets of Tao-Hong-Si-Wu Decoction in Experimental Middle Cerebral Artery Occlusion. Frontiers in Pharmacology, 9, 1570.

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