Circulating cfDNA from plasma was extracted using the QIAamp Circulating Nucleic Acid kit (Qiagen). Sequencing libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems) according to the manufacturer's instructions. Customized xGen lockdown probes (Integrated DNA Technologies) targeting 520 or 474 cancer-relevant genes were used for hybridization enrichment. Then, the capture reaction was performed with Dynabeads M-270 (Life Technologies) and xGen Lockdown hybridization and wash kit (Integrated DNA Technologies), following the manufacturers' instructions. Captured libraries were on-beads PCR amplified with Illumina p5 (5′ AAT GAT ACG GCG ACC ACC GA 3′) and p7 primers (5′ CAA GCA GAA GAC GGC ATA CGA GAT 3′) in KAPA HiFi HotStart ReadyMix (KAPA Biosystems), followed by purification using Agencourt AMPure XP beads. Libraries were quantified by qPCR using the KAPA Library Quantification kit (KAPA Biosystems). Library fragment size was determined by Bioanalyzer 2100 (Agilent Technologies). According to the manufacturer's instruction, the target-enriched library was then sequenced on the HiSeq4000 NGS platform (Illumina). The mean coverage depth was 143× for the whole blood control samples and 4000× for cfDNA samples.
Xgen lockdown hybridization and wash kit
Manufactured by Integrated DNA Technologies
Sourced in United States
The XGen Lockdown hybridization and wash kit is a laboratory product designed for use in targeted sequencing applications. The kit provides reagents and protocols for performing hybridization capture and wash steps to enrich specific genomic regions of interest prior to next-generation sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads m 270, by Thermo Fisher Scientific (6 mentions)
Kapa hyper prep kit, by Roche (6 mentions)
Kapa hifi hotstart readymix, by Roche (5 mentions)
2100 bioanalyzer, by Agilent Technologies (5 mentions)
Kapa library quantification kit, by Roche (5 mentions)
Xgen lockdown probes, by Integrated DNA Technologies (4 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (4 mentions)
Ampure xp beads, by Roche (3 mentions)
Dneasy blood and tissue kit, by Qiagen (3 mentions)
Hiseq4000 ngs platform, by Illumina (2 mentions)
Hiseq 4000 ngs platforms, by Illumina (2 mentions)
Qiaamp circulating nucleic acid kit, by Qiagen (1 mentions)
Idt xgen exome research panel v1, by Integrated DNA Technologies (1 mentions)
Qubit 3.0 with dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
7 protocols using xgen lockdown hybridization and wash kit
1
Targeted Sequencing of Circulating Cell-Free DNA
2
Comprehensive Genomic DNA Extraction and Sequencing
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Genomic DNAs from FFPE samples and the whole blood control samples were extracted using Qiagen QIAamp DNA FFPE Tissue Kit and DNeasy Blood and tissue kits (Qiagen, USA)), respectively, and quantified using Qubit 3.0 with dsDNA HS Assay Kit (ThermoFisher Scientific, USA). Sequencing library preparation was performed with KAPA Hyper Prep Kit (KAPA Biosystems, USA). DNA libraries were pooled and captured with a custom 425 cancer-gene panel. The capture reaction was performed with Dynabeads M- 270 (Life Technologies, CA, USA) and xGen Lockdown hybridization and wash kit (Integrated DNA Technologies) according to manufacturers’ protocols. Captured libraries were PCR amplified with KAPA HiFi HotStartReadyMix (KAPA Biosystems), followed by purification using AgencourtAMPure XP beads. Libraries were quantified by qPCR using KAPA Library Quantification kit (KAPA Biosystems). Library fragment size was determined by Bioanalyzer 2100 (Agilent Technologies). The target-enriched library was then sequenced on HiSeq4000 NGS platforms (Illumina) to a minimum coverage depth of 100X and 600X for blood and FFPE, respectively. Exome capture was performed using the IDT xGen Exome Research Panel V1.0 (Integrated DNA Technologies) and sequenced using HiSeq4000 to a mean coverage depth of ~60X for the normal control (white blood cells samples) and ~150X for the tumor FFPE samples.
3
Cancer Gene Panel Sequencing from FFPE and Blood
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Genomic DNAs from FFPE samples and whole blood control samples were extracted with QIAamp DNA FFPE Tissue Kit and DNeasy Blood and tissue kit (Qiagen), respectively, and quantified by Qubit 3.0 using the dsDNA HS Assay Kit (ThermoFisher Scientific). Library preparations were performed with KAPA Hyper Prep Kit (KAPA Biosystems). A customized panel targeting 425 cancer‐relevant genes was used for hybridization enrichment (Appendix [Link] , [Link] ). The capture reaction was performed with Dynabeads M‐270 (Life Technologies) and xGen Lockdown hybridization and wash kit (Integrated DNA Technologies) according to manufacturers’ protocols. Captured libraries were on‐beads PCR amplified with Illumina p5 (5’ AAT GAT ACG GCG ACC ACC GA 3’) and p7 primers (5’ CAA GCA GAA GAC GGC ATA CGA GAT 3’) in KAPA HiFi HotStart ReadyMix (KAPA Biosystems), followed by purification using Agencourt AMPure XP beads. Libraries were quantified by qPCR using KAPA Library Quantification kit (KAPA Biosystems). Library fragment size was determined by Bioanalyzer 2100 (Agilent Technologies). The target‐enriched library was then sequenced on HiSeq4000 NGS platforms (Illumina) according to the manufacturer's instructions.
4
Cancer Genomic Profiling Protocol
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Sample processing and genomic profiling were performed in a Clinical Laboratory Improvement Amendments (CLIA)- and the College of American Pathologists (CAP)-accredited laboratory (Nanjing Geneseeq Technology Inc., Nanjing, China) as previously described [12 (link),13 (link)]. In brief, genomic DNA from tumor specimen and control samples were extracted and quantified by Qubit 3.0. Library preparations were performed with KAPA Hyper Prep Kit (KAPA Biosystems, Wilmington, MA). Target enrichment was performed using customized xGen lockdown probes (Integrated DNA Technologies, Coralville, IA) targeting 474 cancer- and radiotherapy response-relevant genes (Radiotron gene panel, Nanjing Geneseeq Technology Inc.) (S2 Table ). The hybridization capture reaction was performed with Dynabeads M-279 (Life Technologies, San Diego, CA) and xGen Lockdown hybridization and wash kit (Integrated DNA Technologies) according to manufacturer’s protocols. Captured libraries were on-beads polymerase chain reaction (PCR) amplified with Illumina p5 and p7 primers in KAPA HiFi HotStart ReadyMix (KAPA Biosystems), followed by purification using Agencourt AMPure XP beads. Libraries were quantified by quantitative real-time PCR using KAPA Library Quantification kit (KAPA Biosystems). Library fragment size was determined by Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).
5
Cancer-Related Gene Enrichment Sequencing
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Genomic DNA from FFPE sections or biopsy samples and whole blood control samples were extracted using the QIAamp DNA FFPE Tissue kit and DNeasy Blood and Tissue Kit (Qiagen, USA), respectively. Sequencing libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems) according to the manufacturer’s instructions for different sample types. Customized xGen Lockdown Probes (Integrated DNA Technologies) targeting 437 cancer-related genes were used for hybridization enrichment. The capture reaction was performed with Dynabeads M-270 (Life Technologies) and xGen Lockdown Hybridization and Wash Kit (Integrated DNA Technologies) according to the manufacturer’s protocols. Captured libraries were constructed by on-beads PCR amplification with Illumina p5 (5’-AAT GAT ACG GCG ACC ACC GA-3’) and p7 primers (5’-CAA GCA GAA GAC GGC ATA CGA GAT-3’) on the KAPA HiFi HotStart ReadyMix (KAPA Biosystems), followed by purification using Agencourt AMPure XP beads. Libraries were quantified by quantitative PCR using the KAPA Library Quantification Kit (KAPA Biosystems). Library fragment size was determined by Bioanalyzer 2100 (Agilent Technologies). The target-enriched library was then sequenced on the HiSeq4000 NGS platform (Illumina) according to the manufacturer’s instructions. The mean coverage depth was 317X for the whole blood control samples and 1320X for the tumor samples.
6
Targeted NGS of FFPE Tissue Samples
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Genomic DNA from formalin-fixed paraffin-embedded tissue sections was extracted with QIAamp DNA FFPE Tissue kit (Qiagen). Sequencing libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems) according to manufacturer’s instructions for different sample types. Customized xGen lockdown probes (Integrated DNA Technologies) targeting 446 leukemia- and lymphoma-related genes were used for hybridization enrichment. Capture reaction was performed with Dynabeads M-270 (Life Technologies) and xGen Lockdown hybridization and wash kit (Integrated DNA Technologies) according to manufacturers’ protocols. Genomic DNA was extracted for NGS analysis. All samples subjected to NGS analysis were required to have >10% of tumor cells as identified by immunohistochemistry.
7
Targeted Sequencing of 425 Genes for Comprehensive Analysis
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A commercial 425‐gene panel (Nanjing Geneseeq Technology Inc.) that covered all the CDS regions of the 425 genes and part of spanning introns was used for hybridization enrichment. The tested genes were shown in Table S1 . The capture reaction was performed with Dynabeads M‐270 (Life Technologies, Carlsbad, CA, USA) and xGen Lockdown hybridization and wash kit (Integrated DNA Technologies, Redwood, CA, USA) according to manufacturers' protocols. Captured libraries were amplified by PCR for 6–8 cycles, purified using AMPure XP beads, and quantified by qPCR (KAPA). Libraries were normalized to 2.5 nm and pooled on the chip. Sequencing was carried out with the HiSeq 4000 (Illumina, San Diego, CA, USA) with 2 × 75‐bp paired‐end reads. The average depth was 300×, 1500×, and 6000× for normal blood controls, FFPE, and cell‐free DNA (cfDNA), respectively.
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