Sequencing the Genomes of Single Cells
Single-cell genome sequencing can detect low-frequency genetic alterations present in complex tissues. However, the experimental procedures are technically challenging. This includes dissociation of the tissue, isolation of single cells, whole-genome ampl
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Introduction Sequencing the genomes of small numbers of immune cells requires whole-genome amplification prior to sequencing library preparation. This may be necessary when working with sorted populations of rare immune cell subsets [1], immune cells infiltrating tissues such as tumors [2, 3], or when sequencing the genomes of single normal or malignant immune cells [4]. This process generally requires three steps that are driven by the hypothesis being tested. The first step is isolating the cells of interest. Depending on the number of cells available, as well as the number that will be analyzed, this can be accomplished with manual manipulation, flow-activated cell sorting (FACS), or isolation in a microfluidic device. The second step is amplifying the genomes of the isolated cells. There are now a number of whole-genome amplification methods available, so the method chosen for a given experiment will depend on whether the question requires the detection of large regions of copy number variation (CNV) or smaller regions of nucleotide variation such as single nucleotide variants (SNV) or indels. The final step is to select how the amplification products will be interrogated. This, again, should be tailored to the questions that
Valentina Proserpio (ed.), Single Cell Methods: Sequencing and Proteomics, Methods in Molecular Biology, vol. 1979, https://doi.org/10.1007/978-1-4939-9240-9_14, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Veronica Gonzalez-Pena and Charles Gawad
the study is trying to answer. The amplification products generally undergo low-pass whole-genome sequencing for CNV detection. For SNV detection, the samples can undergo whole-genome sequencing, but investigators generally focus on coding regions using target enrichment.
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Materials Cell Isolation
Most methods of cell isolation from solid tissues comprise both mechanical and enzymatic dissociation. Specific enzyme combinations and dissociation times should be optimized and selected based on optimal viable cell yield and representation of expected cell populations or cell type of interest. 1. For cryopreserved suspension cells: ThawStar automated cellthawing system. 2. For solid tissue cells: Tissue dissociation enzymes or Miltenyi tissue dissociation kit and heated shaker or gentleMACS Dissociator System. 3. PluriStrainer cell strainer (PluriSelect). 4. Washing buffer (PBS with 1% BSA). 5. Fluorescence-based cell counter (Luna FL cell counter or similar). 6. Single cell isolation device (Micromanipulator, FACS sorter, microfluidic device).
2.2 Whole-Genome Amplification (WGA)
The method chosen to amplify the genomes of single cells depends on whether the experiment is designed to detect CNV or SNV. There are a number of different technologies on the market, but we have included the kits that we recommend for each of these applications. 1. For evaluating CNV, we recommend a PCR-based wholegenome amplification method such as DOP-PCR from Sigma or PicoPlex from Takara. 2. For detecting SNV or indels
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