ChIP was performed as mentioned in (for details, see the Supplemental Material ; Asano et al. 2009 (link)). Immunoprecipitated DNA was purified using a ChIP DNA clean and concentrator kit (Zymo Research). ChIP-seq libraries were prepared from 3–5 ng of ChIP-enriched DNA using KAPA Hyper preparation kit (Kapa Biosystems) and sequenced using a HiSeq 2500 (Illumina). Bioinformatics details are included in the Supplemental Material .
Kapa hyper preparation kit
Manufactured by Roche
Sourced in United States
The KAPA Hyper Preparation Kit is a laboratory equipment product designed for DNA library preparation. It provides the necessary reagents and enzymes to prepare DNA samples for next-generation sequencing applications.
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Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Dna 1000 kit, by Agilent Technologies (4 mentions)
Qubit dna dsdna assay kit, by Thermo Fisher Scientific (3 mentions)
Ampure xp bead, by Beckman Coulter (3 mentions)
Le220, by Covaris (3 mentions)
Accugreen high sensitivity dsdna quantitation kit, by Biotium (3 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
Qubit dsdna hs high sensitivity assay kit, by Thermo Fisher Scientific (2 mentions)
Hiseq 2500, by Illumina (1 mentions)
Chip dna clean and concentrator kit, by Zymo Research (1 mentions)
M220 ultrasonicator, by Covaris (1 mentions)
2200 tapestation instrument, by Agilent Technologies (1 mentions)
Novaseq 6000 sequencing system, by Illumina (1 mentions)
E220 focused ultrasonicator, by Covaris (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
M220 system, by Covaris (1 mentions)
M220 focused ultrasonicator, by Covaris (1 mentions)
Hiseq x platform, by Illumina (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
11 protocols using kapa hyper preparation kit
1
ChIP-seq Protocol for Transcription Factor Binding
2
cfDNA Library Preparation and Quantification
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For cfDNA samples, 10–50 ng DNA was used for library construction. For the blood cell and FFPE DNA samples, 200–500 ng DNA was sheared into 200-bp fragments with a Covaris M220 ultrasonicator (Covaris, MA, USA) according to the recommended protocol. Libraries were prepared with a KAPA Hyper Preparation kit (Kapa Biosystems, MA, USA). According to the DNA quality prior to PCR, the ligated fragments were amplified for 6–11 cycles. DNA was purified by using Agencourt AMPure XP beads (Beckman-Coulter, CA, USA), and double size selection was conducted during library preparation. The Qubit DNA dsDNA assay kit (Life Technologies, CA, USA) was used to quantify the libraries, and a 2100 bioanalyzer with the DNA 1000 kit (Agilent, CA, USA) was used to determine fragment length.
3
cfDNA Library Preparation Protocol
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For cfDNA samples, 10–50 ng DNA was used for library construction. Extracted DNA (200–500 ng) was sheared into 200-bp fragments with a Covaris M220 Focused-ultrasonicator. Libraries were prepared with KAPA hyper preparation kit (Kapa Biosystems, Wilmington, MA, USA). The ligated fragments were then amplified for 6–11 PCR cycles, and the libraries were purified with agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) for a double size selection. Quality control of the libraries was performed with a Qubit DNA dsDNA assay kit (Thermo Fisher Scientific) a 2100 Bioanalyzer with the DNA 1000 Kit (Agilent, Santa Clara, CA, USA).
4
Whole-Genome Sequencing Library Preparation
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Library preparation: 450 ng of genomic DNA from each sample was fragmented by adaptive focused acoustics (E220 Focused Ultrasonicator, Covaris) to produce an average fragment size of 350 bp. Fragmented DNA was purified using Agencourt AMPure XP beads (Beckman Coulter), and sequencing libraries were generating using the KAPA Hyper preparation kit (KAPA Biosystems) following the manufacturer's instructions using four cycles of amplification. The quality of the library was assessed using a High Sensitivity D1000 kit on a 2200 TapeStation instrument (Agilent Technologies). Sequencing was performed using the NovaSeq 6000 sequencing system (Illumina), generating 150-bp paired-end reads to obtain 10× coverage.
5
FFPE DNA Fragmentation and Library Prep
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Following the recommended parameters, FFPE DNA ranging from 20 to 500 ng was fragmented to approximately 200 bp fragments using the Covaris M220 system. These DNA fragments were subsequently utilized for library construction with the KAPA Hyper Preparation Kit from KAPA Biosystems (USA). Library quantification was performed using the Qubit dsDNA HS (High Sensitivity) assay kit from Thermo Fisher (USA), while library sizes were determined using the Agilent BioAnalyzer 2100 system from Agilent (USA).
6
Genomic DNA Fragmentation and Library Prep
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30 to 300 ng genomic DNA extracted from samples of FFPE and PBL was sheared with Covaris LE220 to the length of 200 bp with recommended settings. Then, fragmented DNA was used to construct library using KAPA Hyper Preparation Kit (Kapa Biosystems, USA, ID: KK8504) according to the manufacturer's instructions. Quantity of libraries was measured using AccuGreen High Sensitivity dsDNA Quantitation Kit (Biotium, USA, ID: Q32854) and size was determined on Agilent Bioanalyzer 2100 (Agilent, USA).
7
Targeted Sequencing of Circulating Cell-Free DNA
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Genomic DNA extracted from white blood cells was fragmented into 200-bp DNA pieces with a M220 Focused-Ultrasonicator (Covaris, Woburn, MA) according to the manufacturer's protocol. Both the cfDNA library and the genomic DNA library were constructed using a KAPA Hyper Preparation Kit (Kapa Biosystems, Wilmington, MA). A 2100 bioanalyzer with a DNA 1000 kit (Agilent, Santa Clara, CA) was used to determine the fragment length. DNA was hybridized to a designed 543-gene panel (Genecast, Wuxi, China) that included major tumor-related genes, covering 1.7 Mb of the genome. The sequencing libraries were quantified using a Qubit dsDNA HS Assay Kit (Cat#Q32854, Thermo Fisher Scientific, Waltham, MA, USA). After library construction, the collected samples were sequenced on an Illumina HiSeq X platform (paired end, 150 bp).
8
Cancer-related Gene Panel Sequencing
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A total of 74.5 ng DNA per tissue sample was isolated to construct the DNA libraries. Firstly, genomic DNA was sheared into 160–200 bp small fragments by the Covaris LE220 (Covaris, New Jersey, USA). Subsequently, a KAPA Hyper Preparation Kit (Kapa Biosystems, Boston, USA) was used to construct a fragmented DNA library. The libraries were quantified with an AccuGreen High Sensitivity dsDNA Quantitation Kit (Biotium, California, USA), and their size was determined by an Agilent Bioanalyzer 2100 (Agilent). The DNA library was captured by a designed panel of 1,406 cancer-related genes (Genecast Biotechnology Co. Ltd., Beijing, China) that are frequently mutated in common solid tumors and span a 2.4 Mb region in the human genome. Finally, the captured library was subjected to NovaSeq 6000 (Illumina) sequencing, which produced paired-end sequencing with the length of each end as 150 bp.
9
Extraction and Preparation of cfDNA
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Genomic DNA from FFPE tumor tissue, white blood cells or cell pellets of pericardial effusion was sheared into 150 to 20 bp fragments using covaris M220 according to the recommended settings. CfDNA and fragmented DNA were input for library construction. Indexed Illumina NGS libraries were prepared using KAPA hyper preparation kit (Kapa Biosystems, United States) according to the manufacturer’s instructions. Ligated fragments were amplified for 9 PCR cycles using indexed primers depending on the DNA mass of pre-PCR. DNA was purified with Agencourt AMPure XP beads (Beckman-Coulter, United States), and dual size selection was performed during the library preparation. All the libraries were quantified with Qubit DNA dsDNA assay kit (Thermo Fisher, United States), and the fragment length was determined on Agilent bioanalyzer 2,100 using the DNA 1000 kit (Agilent, United States).
10
Illumina NGS Library Preparation
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Genomic DNA was sheared with Covaris M220 (Covaris) according to the recommended settings for 150 to 200 bp fragments, and 20 ng of the fragmented DNA was input for library construction. Indexed Illumina NGS libraries were prepared with the KAPA Hyper Preparation Kit (Kapa Biosystems, USA) according to the manufacturer's instructions. The sequence index-ligated fragments were amplified for 9 PCR cycles depending on the DNA mass of pre-PCR. Agencourt AMPure XP beads (Beckman-Coulter, USA) were used to purify DNA and dual size selection during the library preparation. All libraries were quantified by the Qubit DNA dsDNA Assay Kit (ThemoFisher, USA), and the fragment length was determined on the Agilent Bioanalyzer 2100 with the DNA 1000 Kit (Agilent, USA).
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