Chromosome microdissection was carried out as previously described49 (link). Initial DNA amplification of the collected chromosomes was performed using a GenomePlex Single Cell Whole Genome Amplification Kit (WGA4) (Sigma-Aldrich) according to the manufacturer’s protocol. Microdissected DNA probes Mli1 and Mli2 were generated, respectively, from 15 copies of chromosome MLI1 (the largest chromosome) and MLI2 (the largest among the small chromosomes) of M. lignano (Fig. 1a ). Moreover, microdissected DNA probe Mli3_4 was generated from 15 copies of chromosomes MLI3 and MLI4 (note that these two chromosomes are too similar in size to be reliably distinguished on chromosome spreads; see also Results). The PCR products were labeled with Flu- or TAMRA-dUTP (Genetyx, Novosibirsk) in additional 20 PCR cycles using WGA3 kit (Sigma-Aldrich).
Genomeplex single cell whole genome amplification kit wga4
Manufactured by Merck Group
Sourced in United States
The GenomePlex Single Cell Whole Genome Amplification Kit (WGA4) is a product from Merck Group. It is designed to amplify the entire genome of a single cell. The kit provides a uniform and unbiased amplification of the genomic DNA.
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Lab products found in correlation
Wga3 kit, by Merck Group (1 mentions)
Tamra 5 dutp, by Roche (1 mentions)
Giemsa solution, by Merck Group (1 mentions)
Salmon sperm dna, by Thermo Fisher Scientific (1 mentions)
Nebnext ultra 2 dna library prep kit, by New England Biolabs (1 mentions)
Qiaquick pcr purification kit, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometric quantification, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Genechip fluidics station 450, by Thermo Fisher Scientific (1 mentions)
Genechip hybridization oven 640, by Thermo Fisher Scientific (1 mentions)
Genechip scanner 7g, by Thermo Fisher Scientific (1 mentions)
8 protocols using genomeplex single cell whole genome amplification kit wga4
1
Microdissection and Amplification of M. lignano Chromosomes
2
Whole Chromosome Paint Generation from Microdissected Bs
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Whole Chromosome Paints (WCPBs) were derived from two Bs of the A. flavicollis female #24985 (WCP24985aB and WCP24985bB), one of Bs of the male #3727 (WCP3727B), one of Bs of females #3980 (WCP3980B), #3977 (WCP3977B) and #26368 (WCP26368B). Probes were generated earlier by metaphase chromosome microdissection and DNA amplification using a GenomePlex Single Cell Whole Genome Amplification Kit (WGA4) (Sigma-Aldrich, Saint Louis, MO, USA) according to the manufacturer’s protocol [41 (link)], labeled with TAMRA-5-dUTP (Roche) and used for 2D- and 3D-FISH.
3
Characterization of Pale Martin GRC
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DNA probe of pale martin GRC was prepared as described earlier [18 (link)]. The cell suspension was prepared from testicular cells of adult males treated with hypotonic solution (0.88% KCl) at 37 °C for 3 h and then with Carnoy’s solution (methanol: glacial acetic acid, 3:1). It was dropped onto clean, cold, wet coverslips, dried, and stained with 0.1% Giemsa solution (Sigma) for 3–5 min at room temperature. Fifteen copies of GRC contained in round Giemsa-positive bodies located near spermatids were microdissected with a glass needle. DNA was extracted from the microdissected material and amplified with the GenomePlex Single Cell Whole Genome Amplification Kit (WGA4) (Sigma-Aldrich, St. Louis, MO, USA) [27 (link)]. The PCR product was labeled with Flu-dUTP (Genetyx, Novosibirsk, Russia) in additional PCR cycles (Sileks, Moscow, Russia). FISH experiments on the SC spreads were performed according to a standard protocol with salmon sperm DNA (Ambion, Austin, TX, USA) as a DNA carrier [28 ].
4
Microdissected Chromosome MLI1 Library
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We generated the microdissected DNA library from 15 copies of the chromosome MLI1 (DV1 line) as was described previously (Zadesenets et al. 2016 (link); Zadesenets, Schärer et al. 2017 (link)). The initial DNA amplification of the collected chromosomes was performed using a GenomePlex Single Cell Whole Genome Amplification Kit (WGA4) (Sigma-Aldrich, USA) according to the manufacturer's protocol. Preparing the MLI1 DNA library was performed using NEBNext Ultra II DNA Library Prep kit (New England Biolabs, USA) and sequencing on an Illumina MiSeq with paired-end 300 bp reads at the “Molecular and Cellular Biology” core facility of the IMCB SB RAS (Novosibirsk, Russia).
5
Single Cell Whole Genome Amplification
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WGA of single cells was conducted using the WGA4 Genomeplex Single Cell Whole Genome Amplification Kit (Sigma Aldrich Cat#. WGA4). Amplification was carried out according to the manufacturer’s protocol, with the exception of amplification performed with 23 PCR cycles. In short, the cells were lysed by adding 1.5 µl lysis buffer (1:1 solution of 100 mM DTT + 400 mM KOH) to each tube containing a single thawed cell and subsequently incubated for 2 min at 95 °C. A master mix containing 6.5 µl 10 mM Tris-HCl-EDTA pH 8.0 per reaction and 1 µl of the 10x Single Cell Lysis & Fragmentation Buffer was added to the cold reaction. The samples were incubated for 4 min at 99 °C. Successful amplification was confirmed by gel electrophoresis (1.5% agarose gel) with a DNA smear around 200–1200 bp. Further, DNA was purified using a QIAquick PCR Purification Kit (Thermo Fisher, Cat#. K210012 with elution in 50 µl of TE-buffer. Concentration of purified DNA was quantified with Qubit Fluorometric Quantification (Thermo Fisher). Amplified DNA was sheared using sonication (Covaris S2/E210 focused-Ultrasonicater) with the microtube setup and the 200 bp target size protocol for DNA shearing. Final sample volume was 75 µL.
6
Single-Cell Genomic Analysis of Circulating Tumor Cells
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The identified CTCs were subsequently relocated and imaged at 40× for detailed morphometric analysis. To retrieve cells for genomic analysis, an Eppendorf TransferMan NK2 micromanipulator was used to capture the cell of interest in a micropipette and transfer it to a PCR tube containing 2 µL of lysis buffer (200 mM KOH; 50 mM DTT). The lysate was frozen and stored at −80°C for genomic processing. Decontamination of micropipettes and microscope stage was performed 30 min prior to each cell capture procedure.
The lysed cell mixture was thawed and underwent whole-genome amplification (WGA) and sequencing library construction as previously reported (Dago et al. 2014 (link); Thiele et al. 2019 (link)). Briefly, WGA was done using the WGA4 Genomeplex Single Cell Whole-Genome Amplification Kit (Sigma-Aldrich) followed by purification with the QIAquick PCR Purification Kit (QIAGEN). DNA concentration was measured using the Qubit Fluorometer system (Thermo Fisher Scientific), and fragment size distribution was measured with the Agilent 2100 Bioanalyzer (High-sensitivity DNA Kit, Agilent Technologies). Single-cell Illumina sequencing libraries were created using the Illumina paired indexing system, and pools of up to 96 cells were sequenced at the USC Dornsife Sequencing Core to generate ∼500,000 mapped reads per sample (minimum 250,000).
The lysed cell mixture was thawed and underwent whole-genome amplification (WGA) and sequencing library construction as previously reported (Dago et al. 2014 (link); Thiele et al. 2019 (link)). Briefly, WGA was done using the WGA4 Genomeplex Single Cell Whole-Genome Amplification Kit (Sigma-Aldrich) followed by purification with the QIAquick PCR Purification Kit (QIAGEN). DNA concentration was measured using the Qubit Fluorometer system (Thermo Fisher Scientific), and fragment size distribution was measured with the Agilent 2100 Bioanalyzer (High-sensitivity DNA Kit, Agilent Technologies). Single-cell Illumina sequencing libraries were created using the Illumina paired indexing system, and pools of up to 96 cells were sequenced at the USC Dornsife Sequencing Core to generate ∼500,000 mapped reads per sample (minimum 250,000).
7
Targeted Chromosome Fragment Dissection
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The target region of the homologous pair of chromosome 2 was initially dissected under inverted microscopy. The procedure was improved by referring to the methods described in our previous literature (10). In short, eight copies of the region in 2q36 covering the mutation were dissected from G‐banding metaphase spreads of the patients' peripheral blood samples using glass needles. Then, the chromosome fragment was placed in 9 µl of collection fluid in EP tubes, and each whole‐genome amplification (WGA) was performed using a WGA4‐GenomePlex Single Cell Whole Genome Amplification Kit (Sigma‐Aldrich) following the manufacturer's protocol. The WGA products, which were verified covering the mutation sites by PCR using specific primers, were subjected to NGS testing.
8
Whole-Genome Amplification and SNP Genotyping
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Whole-genome amplification (WGA) was performed on the lysate according to the manufacturer’s instructions starting with library preparation and using the WGA4 GenomePlex Single Cell Whole Genome Amplification kit (Sigma-Aldrich). The GeneElute PCR Purification kit was used to purify WGA DNA from each reaction (Sigma-Aldrich). The WGA or genomic DNA was processed for analysis on the 262 K NspI single-nucleotide polymorphism (SNP) genotyping array as recommended by the supplier (Affymetrix Inc.). Hybridization, washing, staining, and scanning were conducted with the GeneChip Hybridization Oven 640, GeneChip Fluidics Station 450, and GeneChip Scanner 7G, respectively, and as recommended by the manufacturer (Affymetrix). Copy number assignments and loss of heterozygosity analysis results were obtained using the Copy Number Analysis Tool 4.0.1 (Affymetrix). The reference data set consisted of 30 normal female genomic DNA samples.
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