Total RNA was isolated from PBMCs using Isospin cell and tissue RNA kit (Nippon Gene) or an RNAdvance v2 kit (Beckman Coulter) according to manufacturer instructions and quantified with an RNA HS Assay Kit (Thermo Fisher) and a Qubit Flex Fluorometer (Thermo Fisher). For transcriptome analysis, 10 ng of RNA were used for library preparation with a QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen) according to the manufacturer’s protocol for low-input RNA samples. To generate single-nucleotide polymorphism (SNP) calls for several donors whose samples were analyzed by scRNA-seq, cDNA libraries were prepared from 500 ng of RNA using a Collibri Stranded RNA Library prep Kit (Thermo Fisher) according to the manufacturer’s protocol for degraded RNA samples. Libraries were quantified with a Qubit 1x dsDNA HS Assay Kit (Thermo Fisher) and a Qubit Flex Fluorometer (Thermo Fisher), and quality was assessed using D1000 ScreenTape and High Sensitivity D5000 ScreenTape with a Tapestation 2200 (Agilent). Pooled libraries were sequenced on a Novaseq 6000 instrument (Illumina) with 1 × 100-bp reads for transcriptome analysis and 2 × 150-bp reads for generation of SNP calls at the Sequencing Section at OIST.
Qubit flex fluorometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Canada
The Qubit Flex Fluorometer is a compact and versatile instrument designed for accurate quantitation of DNA, RNA, and protein samples. It utilizes fluorescence-based detection technology to provide quick and reliable measurements of sample concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000, by Illumina (5 mentions)
Qubit 1x dsdna hs assay kit, by Thermo Fisher Scientific (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (4 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Trueprep dna library prep kit v2, by Illumina (2 mentions)
Direct zol rna miniprep kit, by Zymo Research (2 mentions)
Qiacube, by Qiagen (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
4200 tapestation system, by Agilent Technologies (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Tapestation 4200, by Agilent Technologies (2 mentions)
Nebnext poly a mrna magnetic isolation module, by New England Biolabs (2 mentions)
Rna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Isospin cell and tissue rna kit, by Nippon Gene (1 mentions)
Collibri stranded rna library prep kit, by Thermo Fisher Scientific (1 mentions)
52 protocols using qubit flex fluorometer
1
Transcriptome and SNP Analysis of PBMCs
2
RNA-seq Library Preparation from Purified RNA
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Total RNA was extracted using TRI reagent (BioLabs) according to the manufacturer specifications. RNA concentration was measured using Qubit Flex Fluorometer (ThermoFisher Scientific). RNA sequencing libraries were prepared using the CEL-Seq2 protocol in the Technion genomic Center (TGC), as published by,19 (link) with one modification; instead of single-cells as input, 2 ng purified RNA was taken as input for library preparation. The CEL-Seq2 libraries were analyzed for average fragment size using Agilent 2200 TapeStation (Agilent) and concentration was measured using Qubit Flex Fluorometer (ThermoFisher Scientific). The libraries were sequenced on the Illumina NextSeq 2000 sequencer (Illumina), 12 bases for read 1 and 88 bases for read 2. Demultiplexing was performed in two steps. First, Illumina demultiplexing was performed using bcl2fastq Illumina software with the following parameters: barcode-mismatches =1, minimum-trimmed-read-length = 0, and mask-short-adapter-reads = 0. Second, Cell-seq demultiplexing using the pipeline described in19 (link) was executed with the following parameters: min_bc_quality = 10, bc_length = 6, umi_length = 6, and cut_length = 88. RNA measurements, library preparation and sequencing were performed by the Technion Genomics Center, Technion, Israel.
3
Microbiome Analysis of Fecal Samples
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Fecal microbial DNA was extracted from each sample with the QIAamp PowerFecal Pro Kit (Qiagen, Germany) following the manufacturer’s instructions. The quantity of the extracted DNA was measured using Qubit Flex Fluorometer (Thermo Fisher Scientific, USA). The 16S rRNA gene of the samples was amplified at the V4-V5 hypervariable region by using the 515F (5′-barcoded-CGCTCTTCCGATCTGTGNCAGCMGCCGCGGTRA-3′) forward primer attached to a 5′ Illumina adapter and the 907R (5′-barcode-GTGCTCTTCCGATCCGYCWATTYHTTTRAGTTT-3′) indexed reverse primer. Amplicon sizes and concentrations were measured using an Agilent 2100 bioanalyzer (Agilent Technologies, USA) and Qubit Flex Fluorometer (Thermo Fisher Scientific, USA), respectively. Each amplified 16S rRNA gene library was diluted and pooled together. Sequencing was performed using the Illumina Miseq platform with MiSeq Reagent Kit v2 (300-cycle kits) at the KNU NGS Core Facility (Daegu, South Korea).
4
Comprehensive RNA-seq workflow for CT26 tumors
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CT26 tumor tissues were isolated and total RNA was extracted using the RNeasy 96 QIAcube HT Kit (Qiagen; C/N 74171) according to manufactuers guidelines with a DNase digest included. RNA concentration was determined by Qubit Flex Fluorometer (Invitrogen), RNA purity was determined using a NanoDrop Eight (Thermo Scientific) and RNA integrity was measured using a 4200 Tapestation (Agilent). All samples had a RINe of ≥7.0. Libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs; #E7760L) as per manufactuer’s guidelines with 1000 ng of RNA input. Ribosomal RNA was removed using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England BioLabs; E7490L). Libraries were subjected to 9 cycles of PCR with unique dual indexes (New England BioLabs; E6440L). Libraries were subsequently quantified by Qubit Flex Fluorometer (Invitrogen), and library sizes were determined by 4200 Tapestation (Agilent) and pooled equimolar. Each library was loaded onto one lane of an S4 v1.5 flow cell (300 cycles) (Illumina, #20028312) on an Illumina NovaSeq 6000 at 2 × 150 paired-end configuration.
5
Genotyping of Human Buccal DNA
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Buccal DNA was extracted from collected samples using QIAamp® DNA Investigator kit (QIAGEN, Hilden, Germany), and quantified using Qubit™ 1X dsDNA HS Assay kit with Qubit™ Flex Fluorometer, according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). The genotyping was performed using Affymetrix™ Axiom KORV1.1-96 Array comprising > 827 K SNPs (Affymetrix), according to the manufacturer’s instructions (Thermo Fisher Scientific). Raw SNP data was filtered for quality control (QC) using PLINK v1.9 and SNPolisher (Purcell et al. 2007 (link)), with the criteria of SNPs with call rate > 0.95, Hardy − Weinberg equilibrium p-value > 1 × 10− 4, and minor allele frequency (MAF) cutoffs > 0.1. SNPs on sex chromosomes and duplicated SNPs were excluded. A total of 259,293 SNPs was selected for the subsequent analysis, and haplotypes of these SNPs were estimated using the ShapeIT algorithm (Delaneau et al. 2008 (link)). All experiments were performed in accordance with the relevant guidelines and regulations.
6
Bacterial Genomic DNA Extraction and Sequencing
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Genomic DNA was extracted from pure bacterial colonies using Qiagen’s DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD, USA) on a QIAcube automated workstation (Qiagen). DNA concentrations were determined using the Qubit dsDNA HS Assay Kit on a Qubit flex fluorometer (ThermoFisher Scientific, Waltham, MA, USA). DNA libraries were constructed with the Nextera XT DNA Library Preparation Kit (Illumina®, San Diego, CA, USA) using 0.2 ng/µL of genomic DNA. Resultant libraries were sequenced on an Illumina MiSeq desktop sequencer using v2 sequencing chemistry with 2 × 250 bp pair-end reads per the manufacturer’s protocol. The quality control of raw sequences was performed by FastQC v0.11.9, and the de novo assembly was done using SPAdes v3.14.1. The final contigs of the isolated strain (designated strain 4524) were subjected to BLASTn to identify the reference genome, which shows the highest identity with the contigs. The genome of the 4524 strain was reordered using progressive Mauve with the A. equuli subsp. equuli ATCC 19392 genome as the reference genome [18 (link)]. The FASTA file of the 4524 genomes was used for genome annotation and further analysis.
7
Quantitative PCR for att Site Analysis
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PCR products (Supp. Figure 2) were prepared using QIAquick PCR Purification Kit (Qiagen) and quantitated using Qubit Flex Fluorometer (Thermofisher), as standards for each of the eight att site forms of interest (attB, attP, attL, and attR for both 61icd and 11capE), using the primers listed in Supplementary Table 2 . Genomic DNA samples of 40–60 ng were prepared, along with a hundred-fold dilution series (100 pg, 1.0 pg, 10 fg and 100 ag) of standard curve samples. PCR reactions were performed in PowerUp SYBR Green Master Mix (Applied Biosystems) with each primer at 500 nM using a three-step amplification protocol (58ºC annealing) in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad).
The slope and intercept of each molar standard curve (all r2 values were above 0.98) was taken, and the following formula used to convert from Ct (cycle threshold) value to copy number in the genomic DNA sample: copies = 10(Ct-intercept)/slope. All reaction efficiencies were above 88%. The total copy number of icd regions was taken as: regions = B + (L + R)/2, where B, L and R are the experimental icd attB, attL, and attR counts, respectively. Finally, icd attB and attP counts were reported after normalizing to icd region counts.
The slope and intercept of each molar standard curve (all r2 values were above 0.98) was taken, and the following formula used to convert from Ct (cycle threshold) value to copy number in the genomic DNA sample: copies = 10(Ct-intercept)/slope. All reaction efficiencies were above 88%. The total copy number of icd regions was taken as: regions = B + (L + R)/2, where B, L and R are the experimental icd attB, attL, and attR counts, respectively. Finally, icd attB and attP counts were reported after normalizing to icd region counts.
8
Quantitative Analysis of Gene Expression
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The concentration of genomic DNA was measured with Qubit dsDNA BR assay on Qubit 2.0 Fluorometer or Qubit Flex Fluorometer (Thermo Fisher Scientific). The DNA solution was diluted to a concentration of 0.5 ng/μl prior to qPCR. Each qPCR solution (20 μl) contained 2 μl of DNA (1 ng), 10 μl of TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara), 0.4 μl of ROX Reference Dye, 2 pmol each of the forward and reverse primers. The primers used for qPCR are listed in Supplementary Table S2 . Each qPCR assay was performed in duplicate, using StepOnePlus or QuantStudio3 (Applied Biosystems) according to the manufacturer's instructions. Amplification condition was initial denaturation at 95°C for 30 s followed by 40 times iteration of a 3-step thermal cycle composed of 95°C for 10 s, 55°C for 30 s and 72°C for 5 s. All qPCR runs included 10-fold serial dilutions to generate standard curves. The quantity of CUP1, ENA1 and URA3 was normalized to that of ACT1. The copy number of CUP1 and ENA1 in the standard curves was calibrated by the results of nanopore sequencing.
9
Plasmid DNA Extraction and Purification
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Plasmid DNA extraction was conducted using a previously reported method [20 (link)]. The PureLink HiPure Plasmid DNA Purification Kit (Invitrogen, Carlsbad, CA, USA) was employed, following the manufacturer’s instructions. The extracted plasmid DNA solution was quantified using a Qubit Flex Fluorometer (Thermo Fisher Scientific, Wilmington, DE, USA) based on fluorometry. To verify the integrity of the plasmid DNA, electrophoresis was performed in 2% agarose gel. Subsequently, the samples were stored at −80 C until further use.
10
Viral RNA Sequencing Protocol
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Amplified products were purified using a Wizard SV Gel and PCR Purification Kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Purified products were examined with an Agilent 4200 TapeStation (Agilent Technologies, San Diego, CA, USA) using a D 1000 ScreenTape system. The concentration of each purified product was quantified with a Qubit Flex Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) using a Qubit-iT 1×dsDNA HS assay (Thermo Fisher Scientific). Each sample was diluted to a DNA concentration of 50 ng/µL, and the purified RT-PCR products were used to prepare sequencing libraries using a Nextera Flex DNA sample preparation kit (Illumina, San Diego, CA, USA) before sequencing. All libraries were sequenced on an iSeq 100 platform (Illumina, San Diego, CA, USA). The amplicons were sheared to lengths between 200 and 400 bp, and Illumina sequencing adapters were ligated to the sheared DNA using Nextera Flex (Illumina). The libraries were multiplexed, clustered, and sequenced using a 300-cycle (2 × 150 bp paired-end) ISeq v2 reagent kit (Illumina on the iSeq 100 platform according to the manufacturer’s protocols. NGS was used to obtain the nucleotide sequences of all eight viral segments.
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