Total RNA was prepared from K562 mbIL-21 expanded control and TGFβi NK cells as per manufacturer’s instructions using the Total RNA Purification Plus Kit (Norgen Biotek, Thorold, ON, Canada) and the resulting total RNA was quantified in a Nanodrop ND-1000 spectrophotometer, and checked for purity and integrity in a Bioanalyzer-2100 device (Agilent Technologies Inc., Santa Clara, CA, USA) and submitted to the genomics core at the Nationwide Children’s Hospital for sequencing. Libraries were prepared using the TruSeq RNA Sample Preparation Kit (Illumina Inc., San Diego, CA, USA) according to the protocols recommended by the manufacturer. The quality of the libraries were determined via Agilent 4200 Tapestation using a High Sensitivity D1000 ScreenTape Assay kit and quantified by KAPA qPCR (KAPA BioSystems, Cape Town, South Africa). Approximately 60–80 million paired-end 150 bp sequence reads per library were generated using the Illumina HiSeq4000 platform.
Bioanalyzer 2100 device
Manufactured by Agilent Technologies
Sourced in United States
The Bioanalyzer-2100 is a microfluidics-based electrophoresis system that provides automated, sensitive, and reproducible analysis of DNA, RNA, and protein samples. The device uses lab-on-a-chip technology to perform size-based separation and detection of these biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Agilent Technologies (4 mentions)
Truseq rna sample preparation kit, by Illumina (3 mentions)
Kapa qpcr, by Roche (2 mentions)
Total rna purification plus kit, by Norgen Biotek (2 mentions)
Agilent 4200 tapestation, by Agilent Technologies (2 mentions)
Hiseq 4000 platform, by Illumina (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Rnaqueous total rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Qubit fluorimeter, by Thermo Fisher Scientific (1 mentions)
Quant it dsdna high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions)
Miseq reagent kit v2 reagents, by Illumina (1 mentions)
Agilent dna 1000 kit, by Agilent Technologies (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Nextera xt index kit, by Illumina (1 mentions)
Agencourt ampure xp bead, by Beckman Coulter (1 mentions)
Truseq stranded mrna library prep kit, by Illumina (1 mentions)
Hiseq 2500 sequencer, by Illumina (1 mentions)
Tissueruptor homogenizer, by Qiagen (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Dna 1000 chip, by Agilent Technologies (1 mentions)
13 protocols using bioanalyzer 2100 device
1
RNA Sequencing of Expanded NK Cells
2
Total RNA Isolation from FACS Purified Cells
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Total RNA from FACS purified subsets were isolated using the RNAqueous Total RNA Isolation Kit (Ambion) according to the manufacturer’s instruction. Total RNA was quantified in a Nanodrop ND-1000 spectrophotometer, checked for purity and integrity in a Bioanalyzer-2100 device (Agilent Technologies).
3
Metagenomic Analysis of Bacterial Diversity
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Metagenomic analysis to assess the qualitative and quantitative diversity of bacterial communities was performed using the hypervariable V4 region of the 16S rRNA gene. The sequence of the V4 site is 254 nucleotides and this DNA fragment in each sample was amplified using forward primer Forward515 with the sequence GTGBCAGCMGCCGCGGCGGTAA and reverse primer Reverse806 with the sequence GGACTACHVGGGTWTCTAAT.
Preparation of 16S DNA libraries was conducted using two-step PCR. At the second stage, Nextera XT Index Kit (Illumina, San Diego, CA, USA) was used to attach adapters and indexed barcodes according to the manufacturer’s protocol. After amplification, PCR products using different combinations of specific primers were purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). The concentration of the resulting 16S libraries in solution was determined with a Qubit® fluorimeter (Invitrogen, Waltham, MA, USA) using the Quant-iT™ dsDNA High-Sensitivity Assay Kit. Purified DNA libraries were mixed equimolarly according to the concentrations obtained. The quality of the library prepared for sequencing was assessed on a Bioanalyzer 2100 device (Agilent, Santa Clara, CA, USA) using the Agilent DNA 1000 Kit. The samples were then directly sequenced on a MiSeq instrument (Illumina, San Diego, CA, USA) using MiSeq Reagent Kit v2 reagents (300 cycles).
Preparation of 16S DNA libraries was conducted using two-step PCR. At the second stage, Nextera XT Index Kit (Illumina, San Diego, CA, USA) was used to attach adapters and indexed barcodes according to the manufacturer’s protocol. After amplification, PCR products using different combinations of specific primers were purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). The concentration of the resulting 16S libraries in solution was determined with a Qubit® fluorimeter (Invitrogen, Waltham, MA, USA) using the Quant-iT™ dsDNA High-Sensitivity Assay Kit. Purified DNA libraries were mixed equimolarly according to the concentrations obtained. The quality of the library prepared for sequencing was assessed on a Bioanalyzer 2100 device (Agilent, Santa Clara, CA, USA) using the Agilent DNA 1000 Kit. The samples were then directly sequenced on a MiSeq instrument (Illumina, San Diego, CA, USA) using MiSeq Reagent Kit v2 reagents (300 cycles).
4
RNA-seq Library Preparation from RNAlater-preserved Samples
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Total RNA was extracted from samples preserved in RNAlater, by using the TissueRuptor homogenizer (Qiagen Iberia, Madrid, Spain) and the miRNeasy mini kit (Qiagen Iberia, Madrid, Spain) according to the manufacturer’s instructions. RNA was eluted in RNAse-free water and quantified on a total RNA 6000 Nano series II chip (Agilent, Santa Clara, CA, USA) in an Agilent Bioanalyzer 2100 device. Illumina RNAseq libraries (150–750 bp inserts) were prepared, from 2 mg total RNA, using the TruSeq stranded mRNA library prep kit according to the manufacturer’s instructions (Illumina Inc., San Diego, CA, USA). The quality of the RNAseq libraries was checked using a DNA 1000 chip (Agilent, Santa Clara, CA, USA) and a Bioanalyzer (Agilent 2100, Santa Clara, CA, USA). RNAseq libraries were sequenced on an Illumina HiSeq2500 sequencer, as 2 × 150 nucleotides paired-end reads, according to the manufacturer’s protocol. Image analysis and base calling were performed using the standard Illumina pipeline. The read sequences were deposited in the Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra ); BioProject accession number: PRJNA648859.
5
Circulating Cell-free DNA Isolation
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CfDNA isolation was carried out from 5 ml supernatant of the pleural effusion fluid and/or 5 ml blood plasma samples using QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany). DNA isolation from sediment cell blocks and cytological samples was performed using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Genomic DNA from FFPE tumor biopsy samples was isolated using QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). 50 µl elution buffer served for the DNA dilution. Qubit dsDNA HS Assay Kit was used to determine DNA concentration using a Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, United States). The distribution and fragment size estimation of double stranded cfDNA deriving from effusates was done using a Bioanalyzer 2100 device (Agilent Technologies, Santa Clara, CA, United States).
6
Porcine Muscle RNA Isolation and Sequencing
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Total RNA was isolated from 56 porcine gluteus medius muscle samples (28 HIGH and 28 LOW) by using the acid phenol method implemented in the RiboPure kit (Ambion, Austin, TX). Total RNA was quantified in a Nanodrop ND-1000 spectrophotometer, checked for purity and integrity in a Bioanalyzer-2100 device (Agilent Technologies, Inc., Santa Clara, CA) and submitted to the Centre Nacional d’Anàlisi Genòmica (CNAG, http://www.cnag.cat ) for sequencing. Libraries were prepared using the TruSeq RNA Sample Preparation Kit (Illumina Inc) according to the protocols recommended by the manufacturer. Each library was paired-end sequenced (2 × 75 bp) by using the TruSeq SBS Kit v3-HS, in a HiSeq2000 platform.
7
Robust RNA Quality Assessment for Microarray
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RNA quantity and quality was assessed spectrophotometrically (Nanodrop) and with 6000 Nano chips via a Bioanalyzer 2100 device (Agilent, Santa Clara, CA, USA), respectively. RNA was judged as being suitable for array hybridization only if samples showed intact bands corresponding to the 18S and 28S ribosomal RNA subunits, displayed no chromosomal peaks or RNA degradation products, and had a RIN (RNA integrity number) above 8.0. The Ambion WT Expression kit (Life Technologies, cat. no. 4411974) in conjunction with the Affymetrix GeneChip WT Terminal Labeling kit (Affymetrix, Santa Clara, CA; cat. no. 900671) was used for the preparation of labeled cDNA from 100 ng of total RNA without rRNA reduction. Labeled samples were hybridized on Affymetrix GeneChip Human Gene 1.1 ST arrays that contain 30,000 coding transcripts and over 11,000 long intergenic non-coding transcripts, provided in plate format. Hybridization, washing, and scanning of the array plates was performed on an Affymetrix GeneTitan Instrument, according to the manufacturer's recommendations. Quality control of the hybridizations to the Human Gene 1.1 ST array and primary data analysis were performed according to strict criteria to ensure that the array data were of the highest possible quality.
8
RNA Sequencing of Naive and Expanded NK Cells
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Total RNA was isolated from naive and expanded NK cells by using the Total RNA Purification Plus Kit (Norgen Biotek, Thorold, Ontario, Canada), according to the manufacturer's instructions. Total RNA was quantified in a Nanodrop ND-1000 spectrophotometer, checked for purity and integrity in a Bioanalyzer-2100 device (Agilent Technologies), and submitted to the genomics core at the Nationwide Children's Hospital for sequencing. Libraries were prepared using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, Calif), according to the protocols recommended by the manufacturer. Library qualities were determined by using the Agilent 4200 TapeStation with a High Sensitivity D1000 ScreenTape Assay kit and quantified by using KAPA qPCR (Kapa Biosystems, Wilmington, Mass). Approximately 60 to 80 million paired-end 150 bp sequence reads per library were generated with the Illumina HiSeq4000 platform. Median read length was 142 bp from a median of 80.4 million reads per sample.
9
RNA-Seq Library Preparation and Sequencing
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Before library generation, the quality of the RNAs extracted from each sample was validated using the Agilent 2100 Bioanalyzer device and the RNA 6000 Pico Chip. RNA-seq libraries were prepared with the Illumina TruSeq mRNA Stranded kit from rRNA-depleted RNA samples (Ribo-Zero Magnetic Gold Kit for Human and Drosophila kit, Epicentre) or from poly(A) enriched RNA samples [NEBNext poly(A) mRNA, New England Biolabs]. Deep sequencing was performed using the Illumina HiSeq2000 sequencer (paired-end 50 cycles).
10
Immunoprecipitation and RNA Extraction
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ARC-enriched microdissections were ice-cold homogenized in 0.25 ml homogenization buffer (50 mM Tris, 100 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 200 U/mL Promega RNasin, 1 mg/mL heparin, 100 μg/mL cycloheximide, Sigma protease inhibitor mixture at pH 7.5). After clearing, 20 μL was separated as input and stored at -80°C until further processing. 2 μl of mouse monoclonal anti-HA antibody (HA.11, ascites fluid; Covance) was added to the remaining homogenate and allowed to rotate for 2 h at 4°C. After incubation, 200 μl of Dynabeads protein G magnetic beads (Invitrogen) was added and incubated for 2h at 4°C with rotation. Immunoprecipitates (IPs) were washed 3 times for 10 min with gentle rotation at 4°C in high-salt buffer (50 mM Tris, 300 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 100 μg/mL cycloheximide at pH 7.5). After final wash, samples were placed in a magnet on ice and Qiagen RLT buffer was added to the remaining pellets and input samples. Total RNA was prepared according to manufacturer’s instructions using the RNeasy-plus Mini kit (Qiagen) and quantified with the Quant-iT RiboGreen RNA assay kit (Invitrogen). RNA integrity was assessed on a 2100 Bioanalyzer device (Agilent Technologies) using the RNA 6000 Pico kit (Agilent Technologies).
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