Reagents were obtained and used as received. DNA templates and primers were obtained from Integrated DNA Technologies. The RNA oligonucleotide competitors and precursor RNAs, obtained from Dharmacon, were 2′-ACE-protected (where ACE is 2′-bis(acetoxyethoxy)-methyl ether) and were deprotected according to the manufacturer’s protocol. All RNAs were desalted using PD10 Sephadex (GE Healthcare) columns by first pre-equilibrating the column with 25 ml water, then loading the RNA and eluting with 10 ml water, collecting 1 ml fractions. Autoradiography images were obtained using a Typhoon 9410 variable mode imager (GE Healthcare) and quantified using Quantity One (Bio-Rad) software. All UV-vis measurements for RNA quantification were obtained using a Beckman Coulter DU800 UV-vis spectrophotometer by heating the RNA to 90 °C and measuring the absorption at 260 nm. Complementary DNA (cDNA) samples were quantified using an Agilent Technologies 2100 Bioanalyzer (Model: G1939A) and an Invitrogen Qubit 2.0 Fluorimeter. Subsequent sequencing was carried out on a Life Technologies Ion Proton sequencer with at least 200-fold coverage. EGM-2 Bullet Kit tissue culture medium obtained from Lonza (CC-3162) was used to grow HUVECs directly. All cells were cultured at 37°C in 5% CO2 in 100-mm-diameter dishes unless stated otherwise.
Bioanalyzer 2100
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Bioanalyzer 2100 is a lab instrument designed for the analysis of DNA, RNA, proteins, and cells. It utilizes microfluidic technology to perform electrophoretic separations and provides quantitative and qualitative data on the samples analyzed.
Automatically generated - may contain errors
Lab products found in correlation
2100 bioanalyzer, by Agilent Technologies (15 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (11 mentions)
Novaseq 6000, by Illumina (10 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (8 mentions)
High sensitivity dna chip, by Agilent Technologies (8 mentions)
Qubit high sensitivity dna assay, by Thermo Fisher Scientific (7 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (4 mentions)
Nextera xt index kit, by Illumina (4 mentions)
Taqman probe, by Thermo Fisher Scientific (4 mentions)
Turbo dnase, by Thermo Fisher Scientific (3 mentions)
Nucleospin rna plant kit, by Macherey-Nagel (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Nanodrop, by Thermo Fisher Scientific (3 mentions)
Hiseq 2500 platform, by Illumina (3 mentions)
Quant it hs dsdna reagent kit, by Thermo Fisher Scientific (3 mentions)
Truseq rna sample preparation kit v2, by Illumina (3 mentions)
81 protocols using bioanalyzer 2100
1
RNA Oligonucleotide Purification and Quantification
2
Liver mRNA Sequencing and Differential Expression Analysis
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Messenger RNA (mRNA) from flash‐frozen liver was extracted with the miRNeasy mini kit (Qiagen, Valencia, CA). mRNA were quantified using the Infinite M200 Pro (Tecan, Morrisville, NC) and quality determined with the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Novogene (Sacramento, CA) prepared the sequencing libraries with the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA). Libraries were validated with the 2100 Bioanalyzer and quantified with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and by qRT‐PCR (Applied Biosystems, Foster City, CA). Sequencing was performed on a NovaSeq 6000 (Illumina) with a 2 × 150 paired‐end configuration. Raw sequence data were converted into fastq files and de‐multiplexed using Illumina’s bcl2fastq software. Sequences were quality‐checked using FastQC and aligned to the HG38 reference genome using STAR aligner. HTSeq‐count was used to generate raw counts per gene. Differentially expressed genes between groups were determined using DESeq2. Genes with adjusted P values, or false discovery rates, <0.05 were considered significantly differentially expressed. The data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE181803 .
3
Single-Cell Whole Genome Sequencing of CTCs
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For subsequent CTC single cell WGS, a single CTC from each patient was microdissected using PALM MicroBeam equipment (Zeiss) and placed into a tube. Based on the multiple annealing and looping-based amplification cycles (MALBAC) technique, the single-cell whole genome sequencing kit (Yikon Genomics, China) was used to perform single-cell WGS.19 (link) Followed by sequencing MALBAC at 25x mean sequencing depth, amplified DNA reaches 93% genome coverage ≥1x for a single CTC cell.19 (link) The molecular weight of the amplified DNA product was examined using a Qubit® dsDNA HS Assay kit in a Qubit 3.0 Fluorometer (Life Technologies, USA), and the amplified DNA product’s genomic integrity was confirmed using quantitative PCR (qPCR) using eight randomly chosen loci. DNA samples were deemed to fulfill the further sequencing requirements if their overall mass was more than 2μg and their DNA fragment sizes ranging from 300 bp to 2000 bp.
Following the SureSelectXT Illumina Paired-End Sequencing Library procedure, the library was prepared. The 2100 Bioanalyzer and qPCR using TBS380 picogreen (Invitrogen, USA) were used to evaluate the quality of the library. Illumina’s Next Seq CN500 High-throughput Sequencer has been used to sequence the whole genomes of the captured single cells.
Following the SureSelectXT Illumina Paired-End Sequencing Library procedure, the library was prepared. The 2100 Bioanalyzer and qPCR using TBS380 picogreen (Invitrogen, USA) were used to evaluate the quality of the library. Illumina’s Next Seq CN500 High-throughput Sequencer has been used to sequence the whole genomes of the captured single cells.
4
Atad3 Knockout Mouse RNA-Seq Protocol
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2.5-month-old Atad3 nKO male mice and control littermates were transcardially perfused with ice-cold PBS. Cortex tissues were dissected fast frozen and used for Total RNA extraction (5 biological replicates per group). Following RNA extraction using TRIZOL, samples were treated with Turbo ™ DNase (Invitrogen) and RNA concentration and Quality control RIN (RNA Integrity Number) values were measured using the 2100 Bioanalyzer. RNA-Seq libraries were prepared using Illumina TruSeq Stranded Total RNA Library Prep Gold with a modified RNA fragmentation time of 6 minutes, but otherwise according to manufacturer’s instructions. RNA sample RIN ranged from 6.4–9.1. Input amounts were 250 ng based on sample concentration. All samples were subjected to 10 PCR amplification cycles. Libraries were balanced for pooling using fragment analysis (Agilent 5200) and DNA quantitation (Qubit). The library pool was sequenced on an Illumina NovaSeq 6000 on an S1 flow cell as 2x 100 bp reads. Base calling and demultiplexing was performed in BaseSpace® using default bcl2fastq parameters. RNA Sequencing data was analyzed using Illumina BaseSpace® application tools which included the Illumina TopHat Alignment, Cufflinks Assembly and Differential Expression analysis using DESeq2 normalization. GO term analysis enrichment was done using the DAVID functional annotation tool.
5
Transcriptome Analysis of Plant Leaves
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Seeds from 67 samples were planted and grown under standardized greenhouse conditions for tissue collection. The fourth set of true leaves from each plant was collected and total RNA extracted using NucleoSpin RNA Plant kits (Macherey‐Nagel) following manufacturer's instructions. RNA‐seq libraries were prepared using the Illumina TruSeq Stranded mRNA Library Prep Kit (Illumina, Inc.), with library quality subsequently analyzed by qPCR using KAPA Library Quantification kits (Kapa Biosystems, Inc.), Qubit 2.0 Fluorometric (Invitrogen, Life Technologies), and a 2100 Bioanalyzer. Sample libraries were pooled according to the sequencing instrument's requirements and sequenced using Illumina HiSeq2500.
6
Transcriptome Analysis of Dorsal CA1
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Microdissection of the dorsal CA1 region (modified from ref.55 ) was performed in ice-cold 1x PBS under a binocular (Leica). Samples from both hemispheres were pooled and transferred into RNALater (Ambion). RNA isolation and deep sequencing were performed at the Transcriptome Analysis Lab (TAL, Göttingen, Germany). Total RNA was isolated using TRIzol (Invitrogen) and nucleic acid quantity, quality and purity were determined using a NanoDrop spectrophotometer and a 2100 Bioanalyzer. Library preparation (starting from 1 μg of total RNA), sequencing and raw data analysis was performed as described previously56 (link). Reads were aligned to mouse genome Mus musculus mm10 and counted using FeaturesCount (http://bioinf.wehi.edu.au/featureCounts/ ) as described previously57 (link). PCA plots were created and differential expression, including the p-value adjustment, was performed using DESeq. 2 package of Bioconductor58 (link). Raw data (fastq files) for gene expression in CK-p25 mice was obtained from GSE6515936 (link) and subjected to the same procedures.
7
Transcriptome Profiling of DMRT1 Chickens
8
Arabidopsis Root RNA Extraction Protocol
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Total RNA was extracted from the roots of 21‐day‐old mock‐ and SA190‐colonized Arabidopsis grown on ½ MS with or without 25% PEG with the Nucleospin RNA plant kit (Macherey‐Nagel) following the manufacturer's recommendations. The quality and quantity of the RNA was assessed using Nanodrop‐6000 spectrophotometer, 2100‐Bioanalyzer (RNA integrity number greater than 8.0), and QubitTM 2.0 Fluorometer with the RNA BR assay kit (Invitrogen).
9
Illumina TruSeq Nano DNA Sequencing
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DNA quality and quantity were determined using a 2100 Bioanalyzer and the Invitrogen PicoGreen assay, respectively. Library preparation was performed according to the Illumina TruSeq Nano DNA Sample preparation protocol. The samples were sheared on a Covaris S220 (Covaris, Woburn, MA, USA) to ∼450 bp, following the manufacturer’s recommendation, and each uniquely tagged with one of Illumina’s TruSeq LT DNA barcodes. Sequencing was performed on the Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) resulting in an average of 56.18 million (49.29–77.79 M) 250 bp paired-end reads per sample. Sequence data were deposited in the Sequence Read Archive and are available under the BioProject PRJNA886972 .
10
Transcriptional Profiling of Culex Mosquito
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Total RNA quality was assessed by the HAIB using an Agilent 2100 Bioanalyzer and an Invitrogen Qubit to ensure quality for the libraries for each time point. A total of seven libraries were prepared, one for each time point of 2, 12, 24, 36, 48, 60, and 72 h post-eclosion. The Illumina RNA Sample Prep Kits for mRNA Seq was used to prepare the libraries, which were run using the paired-end 50 nt read module (HAIB), which also conducted the base calling, barcode parsing, and removal of low quality reads. Further cleaning of adapter was performed using Trimmomatic 10 (link). The Cx. quinquefasciatus genome from Vectorbase 11 was used to map the cleaned reads using Tophat 12 (link) and gene expression estimations were performed using Cufflinks, and differential gene expression was tested using Cuffdiff as time series data 13 . After analysis, only genes with expression values ≥1, as measured in number of fragments mapped for per kilo (every thousand) bases of gene length for every million fragments sequenced (FPKM), were retained for expression comparisons 14 . All data have been submitted to the Gene Expression Omnibus at NCBI as accession #GSE51327.
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