rRNA depletion was performed on the extracted total RNAs from tissue and subsequent library prep following the NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) (Cat#7400L, New England BioLabs, England)’s protocol, which depletes both mitochondrial (12S and 16S) and cytoplasmic (5S, 5.8S, 18S, and 28S) rRNA species. cDNA synthesis was performed by using Maxima First Strand cDNA Synthesis Kit for RT-qPCR, with dsDNase (Cat#1671, Thermo Scientific™, USA). End-repair, A tailing, adaptor ligation, and library amplification were performed by using the KAPA HyperPlus kit (Cat#KK8512, Rocha, USA). Completed libraries were quantified by each library by Qubit™ 1X dsDNA High Sensitivity (HS) assay kit (Cat#Q33231, Invitrogen™, USA) and the insert size estimation was measured by TapeStation, using D1000 ScreenTape assay (Cat#5067-5582, Agilent, USA).
Qubit 1x dsdna high sensitivity assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Qubit™ 1X dsDNA High Sensitivity Assay Kit is a fluorescence-based assay designed to quantify double-stranded DNA (dsDNA) samples with high sensitivity. The kit provides reagents and buffers to prepare and measure dsDNA samples using the Qubit 4 Fluorometer.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 4 fluorometer, by Thermo Fisher Scientific (3 mentions)
D1000 screentape assay, by Agilent Technologies (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
High sensitivity dna kit, by Agilent Technologies (2 mentions)
Bioruptor pico, by Diagenode (2 mentions)
Phenol chloroform isoamyl alcohol, by Thermo Fisher Scientific (2 mentions)
Chloroform isoamyl alcohol, by Merck Group (2 mentions)
Maxima first strand cdna synthesis kit for rt qpcr with dsdnase, by Thermo Fisher Scientific (1 mentions)
Nebnext rrna depletion kit v2 human mouse rat, by New England Biolabs (1 mentions)
Smarter universal low input rna kit for sequencing, by Takara Bio (1 mentions)
Qubit 4.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Chromium next gem single cell 3 v3.1 kit, by 10x Genomics (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
Fragment analyzer system, by Agilent Technologies (1 mentions)
High sensitivity ngs fragment analysis kit, by Agilent Technologies (1 mentions)
Cell ranger count, by 10x Genomics (1 mentions)
Chromium next gem chip g single cell kit, by 10x Genomics (1 mentions)
Novaseq sp 100 cycle flow cell, by Illumina (1 mentions)
Bioruptor ucd 200, by Diagenode (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
17 protocols using qubit 1x dsdna high sensitivity assay kit
1
rRNA Depletion and RNA-Seq Library Prep
2
Whole Transcriptome Analysis of cfRNA
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In order to compare the genetic composition of cfRNA before and after surgery, rRNA depletion was not performed in cfRNA as part of the whole transcriptome study (10 (link)).
cfRNAs were converted to the cDNA by using SMARTer® Universal Low Input RNA Kit for Sequencing (Cat#634940, Takara Bio, Japan). End-repair, A tailing, adaptor ligation, and library amplification were performed according to the protocol of the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (Cat# E7645S, New England BioLabs, England). Completed libraries were quantified by each library by Qubit™ 1X dsDNA High Sensitivity (HS) assay kit (Cat#Q33231, Invitrogen™, USA) and the insert size estimation was measured by TapeStation, using D1000 ScreenTape assay (Cat#5067-5582, Agilent, USA).
cfRNAs were converted to the cDNA by using SMARTer® Universal Low Input RNA Kit for Sequencing (Cat#634940, Takara Bio, Japan). End-repair, A tailing, adaptor ligation, and library amplification were performed according to the protocol of the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (Cat# E7645S, New England BioLabs, England). Completed libraries were quantified by each library by Qubit™ 1X dsDNA High Sensitivity (HS) assay kit (Cat#Q33231, Invitrogen™, USA) and the insert size estimation was measured by TapeStation, using D1000 ScreenTape assay (Cat#5067-5582, Agilent, USA).
3
Genomic DNA Extraction for Uncircularized Isolates
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For the four isolates whose sequences could not be circularized or in which the blaCTX-M allelic variant could not be identified after sequencing and assembly, genomic DNA extraction was performed using 12–16 h cultures obtained as mentioned above, using DNeasy Blood & Tissue kit (Qiagen). DNA was eluted in nuclease-free water, and a minimum DNA concentration of 53 ng/µL was obtained, measured using a Qubit 1X dsDNA High Sensitivity assay kit and a Qubit 4.0 fluorometer (Thermo Fisher Scientific).
4
Single-Cell Transcriptome Profiling Protocol
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Single-cell emulsions were generated with the Chromium Next GEM Chip G Single Cell Kit (10X Genomics) and the Chromium Next GEM Single Cell 3′ v3.1 Kit (10X Genomics) following the standard protocol. Libraries were assessed for mass concentration with the Qubit 1X dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific). Library fragment size was assessed with the High Sensitivity NGS Fragment Analysis Kit (Agilent) on the Fragment Analyzer System (Agilent). Libraries were functionally validated with the KAPA Library Quantification Kit (Roche). Initial low-pass “surveillance” sequencing was performed on a NovaSeq SP 100-cycle Flow Cell (Illumina) and data were assessed with the Cell Ranger count (10X Genomics) output.
5
Comprehensive HLA Profiling for Paternity
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In order to check the paternity affiliation in 18 cases with observed mutation events, further analysis of 52 individuals via amplifying 17 linked HLA loci (classical HLA-A, -B, -C, -DRB1/3/4/5, -DQA1, -DQB1, -DPA1, and -DPB1; non-classical HLA-F, -G, -H, and -E genes; and MICA and MICB) at high-resolution (HR) level using MPS assay was performed. Particularly, library preparation was carried out using hybrid capture technology supplied by an AlloSeq Tx17 kit (CareDX, Stockholm, Sweden). The multiplex commercial locus-specific primers were designed to amplify the HLA class Ia (HLA-A, -B, -C) and HLA class Ib (HLA-F, -G, -H, -E) from the 5′ untranslated region (UTR) to the 3′UTR (full gene). The HLA class II genes (DRB1/3/4/5, -DQA1, -DQB1, -DPA1, and -DPB1) were sequenced from exon 1 to the 3′UTR region (full exon to the 3rd field) and the MICA and MICB genes full exon to the 2nd field (Figure 1 ). Libraries were quantified using the Qubit 1X dsDNA High-Sensitivity Assay Kit (Thermo Fisher Scientific, San Francisco, CA, USA). All steps, including target generation, library preparation, clonal amplification, sequencing, and data analysis, were performed according to the manufacturer’s recommendations (Illumina, San Diego, CA, USA).
6
Fragmentation and Library Prep of PhiX174 DNA
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The sequencing libraries were prepared using PCR-free kits from 200 ng of fragmented PhiX174 DNA. For the fragmentation, 1.5 μg of PhiX174 RF II DNA (NEB, Ipswich, MA) was diluted in a final volume of 100 μL TE 10:1 in 0.5 mL Bioruptor microtubes and sonicated using Bioruptor UCD-200 (Diagenode, Seraing, Belgium) at LOW power (160 W) during 12 pulses of 30s ON/90s OFF. A buffer exchange was performed with 10 mL Tris-HCl pH8.0 using the kit NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany). The profile of the fragmented PhiX DNA was controlled on the Agilent Bioanalyzer 2100 instrument using the High Sensitivity DNA kit (Agilent Technologies, Santa Clara, CA), and the average fragment size was determined to be 289 bp. DNA was quantified using the Qubit 1X dsDNA High Sensitivity Assay kit (ThermoFisher Scientific, Waltham, MA) before library preparation.
7
DNA Library Quality Control
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The quality of the DNA libraries was controlled by electrophoresis on a microchip using the High Sensitivity DNA kit (Agilent Technologies, Santa Clara, CA). Electrophoregrams were obtained after migration in the Agilent Bioanalyzer 2100 instrument and after analysis using the Agilent 2100 Expert Software. The total DNA concentration of the libraries was determined on the Qubit 4 fluorometer using the Qubit 1X dsDNA High Sensitivity Assay kit (ThermoFisher Scientific, Waltham, MA). Adapter-ligated DNA fragments were quantified by real-time PCR using the Universal qPCR KAPA SYBR Fast kit (Roche, Basel, Switzerland) before Illumina sequencing.
8
Direct RNA Sequencing from Poly(A) RNAs
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RNA integrity was assessed using Qubit RNA IQ assay kit (Thermo Fisher) before library preparation. 25–50 µg of total RNA was used to make libraries using the direct RNA sequencing kit (Oxford Nanopore Technologies) as previously described with modifications 89 (link). Briefly, after selection of poly(A) RNAs using Oligo d(T)25 Magnetic Beads (New England Bioloabs), 15 pmoles of REL5 adapter (/5Bio/rArArUrGrArUrArCrGrGrCrGrArCrCrArCrCrGrArGrArUrCrUrArCrArCrUrCrUrUrUrCrCrCrUrArCrArCrGrA rCrGrCrUrCrUrUrCrCrGrArUrCrU) was ligated to the 5′ ends of poly(A)-purified RNAs using T4 RNA ligase 1 (New England Bioloabs) for 3 hours at 37°C. 750 ng of REL-ligated poly(A) RNAs was used for library preparation according to manufacturer’s protocol (Oxford Nanopore Technologies). Final libraries were quantified using Qubit 1X dsDNA High Sensitivity (HS) assay kit (Thermo Fisher) and sequenced on a MinION device using R9.4.1 flow cells (Oxford Nanopore Technologies).
9
Extraction and Quantification of cfDNA
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DNA from fresh-froze biopsies was extracted by using the DNeasy Blood & Tissue Kit (Qiagen, 69506) according to manufacturer's instruction. Before extraction, tissue samples were crushed in liquid nitrogen to facilitate lysis.
About 2 ml of venous blood was drawn fasting in the morning the day before surgery from each patient. It was stored in K2 ethylenediaminetetraacetic acid (K2-EDTA) vacutainer. Blood samples were immediately centrifuged at 1600 g for 10 min at 4 °C to remove blood cells. The supernatant was further centrifuged at 16,000 g for 10 min at 4 °C to remove cell debris completely, and about 1 ml plasma was collected and aliquoted into nuclease-free tubes at −80 °C or below.
cfDNA was extracted from plasma using QIAamp Circulating Nucleic Acid Kit (Qiagen, 55114) according to manufacturer's instructions. Tissue DNA and cfDNA were quantified using Qubit 4 Fluorometer (ThermoFisher, Q33226) and the Qubit 1X dsDNA High Sensitivity (HS) Assay Kit (ThermoFisher, Q33231); cfDNA fragment sizes were assessed by LabChip GX Touch Nucleic Acid Analyzer (PerkinElmer) to visualize any contaminating genomic DNA mixed in cfDNA, which were DNA fragments significantly larger than the known average size of the cfDNA (about 160 bp). The quantity of total extracted cfDNA ranged between 10 and 756 ng (average 41.1 ng). Extracted DNA was stored at −20 °C.
About 2 ml of venous blood was drawn fasting in the morning the day before surgery from each patient. It was stored in K2 ethylenediaminetetraacetic acid (K2-EDTA) vacutainer. Blood samples were immediately centrifuged at 1600 g for 10 min at 4 °C to remove blood cells. The supernatant was further centrifuged at 16,000 g for 10 min at 4 °C to remove cell debris completely, and about 1 ml plasma was collected and aliquoted into nuclease-free tubes at −80 °C or below.
cfDNA was extracted from plasma using QIAamp Circulating Nucleic Acid Kit (Qiagen, 55114) according to manufacturer's instructions. Tissue DNA and cfDNA were quantified using Qubit 4 Fluorometer (ThermoFisher, Q33226) and the Qubit 1X dsDNA High Sensitivity (HS) Assay Kit (ThermoFisher, Q33231); cfDNA fragment sizes were assessed by LabChip GX Touch Nucleic Acid Analyzer (PerkinElmer) to visualize any contaminating genomic DNA mixed in cfDNA, which were DNA fragments significantly larger than the known average size of the cfDNA (about 160 bp). The quantity of total extracted cfDNA ranged between 10 and 756 ng (average 41.1 ng). Extracted DNA was stored at −20 °C.
10
Direct RNA Sequencing Library Prep
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Library preparation was performed using the direct RNA sequencing kit (Oxford Nanopore Technologies, SQK-RNA002) as previously described (Ibrahim et al. 2021) with modifications. A minimum of 75 μg of total RNA was used as starting material to purify poly(A) RNAs using Oligo d(T)25 Magnetic Beads (NEB, Cat # s1419S). 50 pmoles of linker (REL5) containing a 5′-Biotin-PC group and a 3’-OH (Sup. Table. 2 ) were ligated to the 5′ end of RNAs using T4 RNA ligase (NEB, Cat #M0204S) for 3 hours at 37°C, as previously described 28 (link). 500–1000 ng of poly(A) RNA was used for library preparation using SQK-RNA002 sequencing kit (Oxford Nanopore Technologies). The final library was quantified using Qubit 1X dsDNA High Sensitivity (HS) assay kit (ThermoFisher # Q33231) and loaded on FLO-MIN106 or FLO-PRO002 flowcells.
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