The SAMMY‐seq experiment was conducted as previously described (Marasca et al, 2016 (link); Sebestyen et al, 2020 (link)). In brief, 4 million AML12 cells were used for one sample. After 5 min treatment of 600 μl CSK buffer at 4°C, the supernatant was collected as S1. The pellets were washed and treated with 10 U Turbo DNase in 200 μl CSK buffer for 1 h at 37°C, the supernatant was collected as S2. Then, the pellets were washed and extracted with 2 M NaCl in CSK buffer for 10 min at 4°C. After centrifugation, the supernatant was collected as S3. The pellets were washed and solubilized in 8 M urea, and this fraction was collected as S4. For DNA sequencing, 50% volume of each fraction was extracted by phenol/chloroform and sonicated into 300–700 bp fragments. The DNA libraries were prepared using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, ND607‐01) followed by next‐generation sequencing (NGS) using the Illumina HiSeq X Ten system. The remaining 50% of each fraction was lysed with SDS loading buffer and prepared for western blotting test.
Vahts universal dna library prep kit for v3
Manufactured by Illumina
Sourced in China, United States
The VAHTS Universal DNA Library Prep Kit for Illumina V3 is a laboratory equipment product designed for the preparation of DNA libraries for Illumina sequencing platforms. The kit provides the necessary reagents and protocols to enable the construction of high-quality DNA libraries from a variety of input DNA samples.
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Lab products found in correlation
Novaseq 6000 sequencer, by Illumina (6 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (6 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (4 mentions)
Novaseq 6000, by Illumina (3 mentions)
Bioruptor pico, by Diagenode (3 mentions)
Hiseq 4000 sequencer, by Illumina (3 mentions)
Sureselect human all exon v7, by Agilent Technologies (3 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (3 mentions)
Hiseq x, by Illumina (3 mentions)
Hiseq 4000, by Illumina (3 mentions)
Ab4729, by Abcam (2 mentions)
Hiseq x ten, by Illumina (2 mentions)
Qsep100 dna fragment analyzer, by Bioptic (2 mentions)
Vahts dna clean beads, by Vazyme (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Hiseq x ten platform, by Illumina (2 mentions)
Sureselect clinical research exome v2, by Agilent Technologies (2 mentions)
Hiseq x ten system, by Illumina (1 mentions)
Qiaamp circulating nuclear acid kit, by Qiagen (1 mentions)
Nebnext ultra 2 q5 master mix, by New England Biolabs (1 mentions)
44 protocols using vahts universal dna library prep kit for v3
1
SAMMY-seq: Chromatin Fractionation Protocol
2
Genomic Analysis of Carbapenem-Resistant K. pneumoniae
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The DNA of each CRKP isolate was purified and recovered by a silica gel column (D3146, HiPure Bacterial DNA kit) after incubation. Paired-end libraries with insert sizes of ~300 bp were prepared following Illumina’s standard genomic DNA library preparation procedure (VAHTS Universal DNA Library Prep kit for Illumina V3) and sequenced on an Illumina NovaSeq 6000 using the S4 reagent kits (v1.5) according to the manufacturer’s instructions. The sequencing reads of each isolate were quality trimmed and assembled into contigs using EToKi (45 (link)). The ST, antibiotic resistance genes, and virulence determinants of each isolate were predicted based on its genomic assemblies using kleborate (46 (link)). All the isolates from ST11 were aligned using the EToKi align module onto a reference genome (GCF_011066505.1 ; sample from blood, Hong Kong, China, 2016) to obtain a multiple sequence alignment of the nonrepetitive, core genomic regions that were shared by ≥95% of genomes. The resulting alignments were subjected to a maximum-likelihood phylogeny by the EToKi phylo module, had the recombinant regions removed using RecHMM (47 (link)), and were visualized using GrapeTree online software (48 (link)). Similarly, we also aligned all the ST14 isolates onto a reference genome (GCF_001521895.1 , sample from urine, Jiangxi, China, 2014) and estimated a maximum-likelihood phylogeny of ST14.
3
Plasma Cell-free DNA Sequencing for MRD
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Cell‐free DNA (cfDNA) was isolated from plasma using the QIAamp Circulating Nuclear Acid Kit (QIAGEN, Venlo, Netherlands). Multiplex PCR was performed using a NEBNext Ultra II Q5 Master Mix (New England Biolabs, Inc., MA, USA). A library was constructed using the VAHTS® Universal DNA Library Prep Kit for Illumina V3 (Nanjing Vazyme Biotech Co. Ltd., Nanjing, China) and sequenced using a NovaSeq 6000 sequencer (Illumina Inc., CA, USA). For a SNP pool comprised of all mutations, two or more mutations were judged to be MRD positive, whereas one or no mutations were judged to be MRD negative.
4
Chromatin Immunoprecipitation of BirA in Clostridia
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The pMTL83151‐P1440‐birA‐3×FLAG plasmid carrying the 3×FLAG‐tagged birA was transformed into the Clju ∆birA strain. Cells were cross‐linked with 1% formaldehyde for 10 min at room temperature. The reaction was quenched by adding glycine to a final concentration of 0.125 M. Formaldehyde cross‐linked cells were washed with pre‐chilled phosphate buffer saline and then sonicated to generate 200–500 bp DNA fragments. Immunoprecipitation was performed using monoclonal anti‐3×FLAG antibody (Cat#F1804; Sigma) and protein A magnetic beads (Cat#10002D; Invitrogen) according to the manufacturer’s instructions. Sonicated extracts, without the addition of antibodies, was performed as a negative control. Proteins were then removed by incubation with proteinase K for 2 h at 55°C. The libraries were prepared using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Catalog NO. ND607; Vazyme) and sequenced using the Novaseq 6000 system (Illumina, San Diego, CA, USA).
5
ALKBH1 Chromatin Immunoprecipitation Sequencing
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Mouse primary hepatocytes were isolated and transfected with plasmids expressing ALKBH1 protein with a flag tag for 48 hours. Then, ChIP experiments were performed using a SimpleChIP Enzymatic Chromatin IP Kit (9003; Cell Signaling) with an antibody against flag (14793; Cell Signaling) or the control normal rabbit IgG, according to the manufacturer’s procedures. DNA sequencing libraries were prepared using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (ND607; Vazyme, Nanjing, China). High-throughput sequencing and analysis were conducted by Seqhealth Technology Co, Ltd (Wuhan, China). Briefly, the libraries were sequenced on a Novaseq 6000 sequencer (Illumina) with the PE150 model. Raw data were filtered by Trimmomatic (version 0.36). Then, clean reads were mapped to the mouse genome (GRCm38) using STAR software (version 2.5.3a). RSeQC (version 2.6) was used for read distribution analysis. MACS2 software (version 2.1.1) was used for peak calling. Bedtools (version 2.25.0) was used for peak annotation and peak distribution analysis. The differentially binding peaks were identified by a python script, using the Fisher test. A P value less than .05 was judged statistically significant enrichment.
6
Plasma cfDNA Extraction and WGS Protocol
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We performed plasma sample collection, cfDNA extraction followed by WGS, as described in Supplementary Materials and Methods. Briefly, the venous blood samples were collected during routine physical checks (healthy volunteers) or on the day of therapy, prior to the first treatment (cancer patients). All samples were collected, shipped, and processed uniformly. A total of 5–10 ng of plasma cfDNA per sample was subject to PCR-free WGS library construction with the VAHTS® Universal DNA Library Prep Kit for Illumina V3 (Vazyme). The libraries underwent paired-end sequencing on DNBSEQ-T7. To minimize bias, the sample operating team was blinded to the case or control status of the samples during the whole process.
7
Genomic Analysis of Emerging Mosquito-borne Virus
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The total RNA of each qRT-PCR-positive mosquito pool was extracted, and reverse transcription PCR (RT-PCR) was performed with random hexamers to generate cDNA. After the cDNA was purified via ethanol precipitation, the sequencing library was constructed using a VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, Nanjing, China), and the quality of the library was inspected using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). The sequencing library was sequenced using an Illumina MiSeq system. The sequence data that were obtained via next-generation sequencing were edited and assembled using the MEGAHIT software package (version 1.2.9) to obtain the whole-genome sequence of HMV2.
In addition, the qRT-PCR-positive samples were also sequenced after nested PCR amplification. Nested PCR was performed, using HMV2-specific primers, to amplify the positive samples (Table 1 ). The nested PCR amplification results were purified via 1% agarose gel electrophoresis and were visualized using an imaging system (ChemiDoc Touch, USA). High-quality amplification products were sent to the Shanghai Sangon Biotech Company for Sanger sequencing.
In addition, the qRT-PCR-positive samples were also sequenced after nested PCR amplification. Nested PCR was performed, using HMV2-specific primers, to amplify the positive samples (
8
Chromatin Immunoprecipitation Sequencing Protocol
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The chromatin immunoprecipitation (ChIP) assay was performed as previously described.27 H3K27ac (ab4729; Abcam), H3K27me3 (9733S; CST), CTCF (07–729; Millipore), H3K9ac (9649; CST), H3K4me (5326; CST) and EP300 (ab275378; Abcam) were used for ChIP experiments. For ChIP‐sequencing (ChIP‐seq), 10–50 ng product was used to generate the DNA library using a VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme Biotech). The ChIP DNA Library was sequenced with Illumina HiSeq X Ten. For ChIP‐qPCR, ChIP products that were used for RT‐PCR were used to amplify the PCR products for 45 cycles.
9
EM-seq Library Preparation for DNA Methylation Analysis
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EM-seq library preparation was performed using VAHTS Universal DNA library Prep Kit for Illumina V3 (Vazyme) and EM-seq Conversion Module (New England BioLabs) according to the manufacturer’s instruction with minor modifications. In brief, mechanically fragmented methylation control DNA (CpG methylated pUC19 and unmethylated λ DNA) alone or combined with cell-free DNA fragments was treated with VAHTS Universal DNA library Prep Kit (New England BioLabs) for end-repair, A-tailing, and ligation of EM-seq adaptor (New England BioLabs). The ligated samples were methyl-converted with EM-seq Conversion Module (New England BioLabs) per the manufacturer’s protocol. Methyl-converted DNA was purified and amplified using NEBNext Unique Dual Index Primers (New England BioLabs) and KAPA HiFi HotStart Uracil + ReadyMix (KAPA biosystems).
10
High-quality Genome Sequencing of F. longipetiolata
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Leaves were taken from the F. longipetiolata seedlings cultivated at Guizhou University, Guizhou Province, China (26°4.504′ N, 106°6.568′ E), and lodged a voucher specimen (accession number FL-GZU-001) in the Institute for Forest Resources & Environment of Guizhou at Guizhou.
A Plant Genomic DNA Kit (TIANGEN, Beijing, China) was used to extract total genomic DNA from 100 mg of the leaves. The purified DNA was then fragmented by mechanical disruption (sonication). Then, the paired-end (PE) library was constructed using VAHTS Multiplex Oligos set 4 for Illumina (Vazyme, Nanjing, China) and VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, Nanjing, China), according to the manufacturer’s protocols. Finally, the qualified libraries were sequenced on the Illumina platform, according to the paired-end PE150 sequencing strategy. Approximately 6 Gb of raw data were sequenced. All of the above works were conducted by Genepioneer Biotechnologies Co. Ltd. (Nanjing, China).
A Plant Genomic DNA Kit (TIANGEN, Beijing, China) was used to extract total genomic DNA from 100 mg of the leaves. The purified DNA was then fragmented by mechanical disruption (sonication). Then, the paired-end (PE) library was constructed using VAHTS Multiplex Oligos set 4 for Illumina (Vazyme, Nanjing, China) and VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, Nanjing, China), according to the manufacturer’s protocols. Finally, the qualified libraries were sequenced on the Illumina platform, according to the paired-end PE150 sequencing strategy. Approximately 6 Gb of raw data were sequenced. All of the above works were conducted by Genepioneer Biotechnologies Co. Ltd. (Nanjing, China).
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