RNA was extracted and purified from the Ptm lymphocytes using the RNeasy Mini kit (Qiagen) following manufacturer's protocol. Total RNA integrity was checked using an Agilent 2200 TapeStation (Agilent Technologies) and quantified using a Trinean DropSense96 spectrophotometer (Caliper Life Sciences). RNA-seq libraries were prepared from total RNA using the TruSeq RNA Sample Prep Kit v2 (Illumina) and a Sciclone NGSx Workstation (PerkinElmer). Library size distributions were validated using an Agilent 2200 TapeStation (Agilent Technologies). Additional library QC, blending of pooled indexed libraries, and cluster optimization were performed using Life Technologies’ Invitrogen Qubit 2.0 Fluorometer (Life Technologies-Invitrogen). RNA-seq libraries were pooled (6-plex) onto a flow cell lane. Sequencing was performed using an Illumina HiSeq 2500 in rapid mode employing a paired-end, 50 base read length (PE50) sequencing strategy. Image analysis and base calling were performed using Illumina's Real Time Analysis v1.18 software, followed by 'demultiplexing' of indexed reads and generation of FASTQ files, using Illumina's bcl2fastq Conversion Software v1.8.4.
Invitrogen qubit 2.0 fluorometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Invitrogen Qubit 2.0 Fluorometer is a compact and accurate instrument designed for the quantification of DNA, RNA, and protein samples. It utilizes fluorescent dye-based assays to provide precise and sensitive measurements of these biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (3 mentions)
Hiseq 2500, by Illumina (2 mentions)
Sciclone ngsx workstation, by PerkinElmer (1 mentions)
Real time analysis v1, by Illumina (1 mentions)
Bcl2fastq conversion software v1, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Truseq rna sample prep kit v2, by Illumina (1 mentions)
Agilent 2200 tapestation, by Agilent Technologies (1 mentions)
Nextseq 2000, by Illumina (1 mentions)
Agilent 4200 tapestation, by Agilent Technologies (1 mentions)
Truseq stranded mrna kit, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Bioanalyzer high sensitivity dna kit, by Agilent Technologies (1 mentions)
8 protocols using invitrogen qubit 2.0 fluorometer
1
RNA-seq Library Preparation and Sequencing
2
Liver Transcriptome Profiling in Nrf2 Mice
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Total RNA derived from the livers of wild-type, C151S, and Nrf2-knockout mice treated with vehicle (n = 5) and CDDO-Me (n = 5) in each genotype were used. RNA quality check, library construction, and sequencing were performed at the Fred Hutchinson Cancer Center Shared Resources Cores for Genomics and Bioinformatics. Total RNA integrity was affirmed and quantified using an Agilent 4200 TapeStation (Agilent Technologies, Inc., Santa Clara, CA) and Trinean DropSense96 spectrophotometer (Caliper Life Sciences, Hopkinton, MA), respectively. RNA-seq libraries were prepared from total RNA (TruSeq Stranded mRNA kit, Illumina, Inc., San Diego, CA), and library size distribution was validated (Agilent 4200 TapeStation, Agilent Technologies, Santa Clara, CA). Additional quality control of the library, combining of pooled indexed libraries, and cluster optimization was performed using Life Technologies’ Invitrogen Qubit 2.0 Fluorometer (Life Technologies-Invitrogen, Carlsbad, CA). RNA-seq libraries were pooled (30-plex) and clustered onto a P3 flow cell. Samples were sequenced using an Illumina NextSEq 2000 using a paired-end, 50 base read-length strategy. Raw data have been deposited in Gene Expression Omnibus; the accession number is GSE222256.
3
Genomic DNA Isolation and Quantification
4
Extraction and Quantification of Total RNA
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Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion, USA) according to the manufacturer’s protocol. Briefly, hippocampal tissues from male and female rat pups (n = 3 pups/sex/treatment group from three different dams) were obtained and lysed in denaturing lysis buffer, which stabilized RNA and inactivated RNases. Total RNA was then extracted from the hippocampal tissue lysates using acid–phenol:chloroform and further purified over a glass–fiber filter. RNA was eluted from the purification column using the elution solution. The purity of total RNA was assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) and quantified using an Invitrogen Qubit 2.0 Fluorometer (Thermo Fisher Scientific, USA).
5
Quantifying M. tuberculosis CFUs
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We measured the DNA concentration of the thermolysates through an Invitrogen Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. The calculated weight of DNA per M tuberculosis bacillus served as the divisor for each DNA concentration, the quotient of which was the equivalent colony-forming units (CFUs) per milliliter of each thermolysate.
We subjected thermolysates with initial concentrations of 108 to 109CFU/mL to 1:2 and 10-fold serial dilutions until we obtained 106 to 107CFU/mL, respectively. We then dispensed a 1:2 mixture of diluted thermolysate and sample reagent in the Xpert MTB/RIF cartridge following the manufacturer’s instructions.
We subjected thermolysates with initial concentrations of 108 to 109CFU/mL to 1:2 and 10-fold serial dilutions until we obtained 106 to 107CFU/mL, respectively. We then dispensed a 1:2 mixture of diluted thermolysate and sample reagent in the Xpert MTB/RIF cartridge following the manufacturer’s instructions.
6
Isolation and Quality Assessment of Total RNA
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Total RNA was extracted and purified using the mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Briefly, prefrontal cortex tissues were lysed in a denaturing lysis buffer, which stabilized RNA and inactivated RNases. Prefrontal cortex tissue lysates were then subjected to acid-phenol:chloroform extraction to purify RNA and remove DNA. Ethanol was then added to the samples and passed through a filter cartridge containing a glass-fiber filter that immobilized the RNA. The filter was then washed three times, and finally, total RNA was eluted with a low ionic-strength solution. The purity of total RNA was assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and quantified by using Invitrogen Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). An Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to determine RNA integrity. The results from the bioanalyzer were presented as the 28S:18S rRNA ratio and the RNA integrity number (RIN). The 28S:18S rRNA ratio was greater than 1.0, and the RIN was greater than 7.0 for all RNA samples.
7
Extraction and Purification of Total RNA from Rat Prefrontal Cortex
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Total RNA was extracted from the prefrontal cortex of rat pups and purified using the mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. The protocol was described in our previous study [36 –38 (link)]. Briefly, prefrontal cortex tissues were lysed in a denaturing lysis buffer to stabilize RNA and inactivate RNases. Then, the lysates were subjected to acid-phenol: chloroform extraction to purify RNA and remove DNA. Ethanol was added to the samples and passed through a glass-fiber filter cartridge that immobilized the RNA. The filter was then washed three times, and finally, total RNA was eluted with a low ionic-strength solution. The purity of total RNA was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) and quantified using an Invitrogen Qubit 2.0 Fluorometer (Thermo Fisher Scientific, USA).
8
Histone ChIP-seq Library Preparation
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Prepared libraries were quantified using the dsDNA HS assay for Invitrogen Qubit 2.0 Fluorometer (ThermoFisher) and size distribution was measured with the Bioanalyzer High Sensitivity DNA Kit (Agilent). DNA libraries resulting from MNase digestion and ChIP were sequenced on an Illumina HiSeq2500 in high output run mode. All histone ChIP-seq reads were first trimmed for adapter sequence and low quality tails (Q < 20) with Trim Galore (v.0.4.2) [35, 42] . We utilized deeptools2 [49] to investigate the quality of replicates (multiBamSummary, plotFingerprint and plotCorrelation tools) with subsequent down sampling of some histone ChIP replicates, which had rather high coverage (see Suppl. Table 1; sheet sequencing depth). All raw read data of this study has been deposited at ENA, accession no. PRJEB46233.
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