This study was funded by the Austrian Science Fund (FWF), project-no. I 1220-B19 (to P.S.) as part of the ERANET project in Translational Cancer Research (TRANSCAN) &#34;Circulating Tumour Cells as Biomarker for Minimal Residual Disease in Prostate Cancer (CTC-SCAN)&#34;. PhD candidate S.C. was trained within the frame of the PhD Program Molecular Medicine of the Medical University of Graz. The authors gratefully acknowledge Nina Schl&#246;gl, Daniel Kummer, Gabi Wendt, Claudia Chudak, Julia Schulz, and Johanna Schiller for their expert technical assistance. The authors thank Georg Peinhaupt for graphical design support.

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The authors have nothing to disclose.

The described protocol aims at retrieving cells from a functionalized wire (referred to as Device) for ex vivo single-cell genomic analysis. We tested three EpCAM-positive cell lines (HT-29, LNCaP, and VCaP), but in principle, this method can be applied to any cell line expressing EpCAM. EpCAM-positive target cells can be presented to the Device as pure cell suspension (see 2.1.) or mixed into a surplus of background cells (e.g. peripheral blood, see 2.2.). Whereas especially the latter might be used to test isolation capacities under rare cell conditions, fine tuning of the number of cells attached to the wire can be done using the described low-volume approach (see 2.3.). As differences between cell lines are to be expected, suitable cell densities for charging the wire, rotation speed as well as incubation time need to be tested empirically by evaluating the number of attached cells using an immunofluorescence microscope.
For genomic downstream applications at the single-cell level WGA, is required. The WGA method of choice may differ between labs and, in principle, our method is open to all methods than can handle samples obtained from micromanipulation or laser microdissection. In this protocol, we use a method based on enzymatically fragmented DNA due to a better performance as compared to a heat-fragmentation based WGA7. Optional, single cells can be forwarded to isothermal WGA allowing subsequent DNA profiling20,21.
The described protocol aims at recovering intact cells after being attached to a new generation of functionalized wires for genomic single-cell analysis. The first generation of functionalized wires, which also represent the only CE-approved in vivo enrichment devices on the market so far, were used for enumerating CTCs in metastasized cancer patients. Previous publications and our own data suggest the in vivo isolation technique to be more sensitive compared to the current gold standard technology2. Thus, equipping this in vivo isolation technique with a recovery feature as realized in the Device allows combining high CTC recovery with characterization at the single-cell level.
However, the Device is not cleared for the use in patients, thus, clinical data are not available. Ex vivo applications (i.e. isolating CTCs from peripheral blood after sampling) are not recommended as the technology is adapted to the settings present in the cubital vain, e.g. low diameter of the vain (increasing the probability of direct contact between CTCs and catching antibodies) and long retention time (30 min, allowing 2-3 L of blood to pass the wire). However, as outlined in section 2.2. target cells can also be isolated from mixed samples7.
A current limitation of the method is the inefficient recovery of unfixed cells after detachment from the wires. This, however, might be improved by a cell fixation step prior to the detachment treatment.
In summary, this protocol is a first step towards the combined use of an in vivo isolation technique with cell recovery for genetically characterizing single cells. Further efforts need to address the optimization of the cell recovery and clearance for its application in patients1,2. However, personalized medicine for treatment monitoring and therapy decisions is beyond enumeration of CTCs. Thus, this method is a first step towards genomic CTC characterization at the single cell level.
Applications towards single-cell transcriptome analysis seem feasible as intact cells are harvested. However, it remains to be assessed whether the recovering procedure has an impact on RNA integrity (e.g. forwarding recovered single cells to direct lysis followed by RT-qPCR22). If successful, characterization of single cells (e.g. CTCs) can be shifted to the transcriptome level , allowing more detailed analyses. In this regard, RNA-seq using Smart-seq2 provides a valuable tool for RNA downstream analysis of single cells as the preamplified cDNA (obtained during RNA-seq library preparation) can be subjected to quantitative PCR. This will allow a combined target and screening-based analysis of single recovered cells22,23.
The current functionalized wires are based on anti-EpCAM antibodies which is a widely used epithelial marker for positive selection of CTCs24. As several CTCs might downregulate epithelial markers such as cytokeratins or EpCAM25, adding antibodies such as HER2/new would increase the chance of CTC isolation. Several antibody based enrichment strategies have been developed and reviewed by Ferreira et al. in 201626. A mixture of antibodies immobilized on a wire could be a future improvement of the technology leading to the isolation of a higher number and additional subtypes of CTCs.
It is mandatory for all steps involving wire handling to keep the functional part of the wire submerged in solution in order to maintain its functionality. We recommend to place the wire in 1x PBS during evaluation (microscope; e.g. steps 3.1. - 3.3.) and storage. In order to avoid inappropriate staining, we recommend using freshly prepared staining solution in steps 1.2.1. and 1.2.2. Weakly stained cells hamper cell counting on the wire and may lead to the underestimation of the attached cells.
It is necessary to avoid plugging the Pasteur pipette with the rubber cap when inserting the wire in the Pasteur pipette for subsequent loading of the cell suspension (step 2.3.4.). The rubber cap is used to hold the wire in place so that the functional part is situated inside the tip of the Pasteur pipette. If the cap is attached too firmly to the rear of the Pasteur pipette, loading of the cell suspension is not possible. In this case, ease the cap such that the air which is displaced by the loaded cell suspension can leave the pipette.
Bending the wire is crucial for its orientation during the cell counting in steps 3.2. and 3.3. Mount the wires using the adhesive tape such that the non-functional end of the wire comes to lie to one side. After scanning of the entire length of the functional part, the wire is rolled 180&#176; so the other half of the functional wire can be scanned. This step is important for initial experiments to assess the number of cells on the wire. Once the number of cells for a certain cell line and procedure is evaluated, the procedure can be repeated without cell counting (e.g. only checking for presence of cells or omitting this step). Careful pipetting is crucial to avoid cell loss when reducing the volume of the supernatant in step 4.8. Make sure pipetting is done smoothly without causing turbulences to the pellet.

Recently, CellCollector DC01, a medical wire functionalized with anti-EpCAM antibodies for isolating CTCs from peripheral blood of cancer patients, was added to the methodical spectrum of CTC enumeration1,2,3. In a currently ongoing study in non-metastatic prostate cancer, this functionalized wire reports almost twice as many patients to be CTC-positive and higher CTC counts in CTC-positive patients as compared to CellSearch, the gold standard in CTC enumeration3. Due to this encouraging performance, isolation of cells from a functionalized medical wire for single-cell analysis would be desirable, but neither enzymatic treatment with cell detachment solutions (e.g. trypsin) nor laser microdissection allows the recovery of intact cells (data not shown).
To allow the detachment of captured cells, a new generation of functionalized wires was equipped with a specific polymer. This polymer, which links the capture antibodies to the wire, is susceptible to a release buffer treatment allowing detachment of intact cells (CellCollector DC03 referred to as Device). The new functionalized device, allows isolation of target cells from various concentrations of cancer cell line cells spiked into bovine serum albumin (BSA)/phosphate buffered saline (PBS) and peripheral blood, respectively.
To ease the visual detection of cells on the Device and after recovery, the target cancer cells are labelled with carboxyfluorescein succinimidyl ester (CFSE) and a DNA stain before the recovering treatment (i.e. charging and detaching). The treatment&#39;s effects on DNA quality of single cells were previously evaluated after WGA by means of a quality control PCR4,5, array-CGH6,7&#160;and targeted sequencing7 showing no difference compared to non-treated cells micromanipulated from cell suspensions7. The advantage of this method lies in the combination of rare target-cell pre-enrichment and the recovery of cells for single-cell downstream analysis (Figure 1). The current CE-labelled in vivo collection device is generally used for enumeration of CTCs rather than for single-cell molecular characterization2,8. However, more comprehensive analysis to investigate heterogeneity among CTCs long for analysis at the individual cell level (i.e. targeted sequencing at the single-cell level). Other cell-based methods are based on immunomagnetic isolation of EpCAM-positive CTCs and single-cell handling based on dielectrophoresis for subsequent molecular genetic analysis9,10. Molecular characterization of CTCs is an important requirement for their useful implementation in a clinical setting and is equally important in basic research of the metastatic cascade. In parallel to CTCs, circulating tumor DNA (ctDNA) has become of great importance as it allows DNA analysis of the tumor burden with minimal technical isolation procedures11,12. The cell based approaches might serve as a complementary contribution as it allows for RNA13,14 and protein15 expression analysis and also for CTC derived cell cultures or xenografts16,17. Although obstacles such as low cell recovery and clearance for the use in patients still need to be overcome, the catch and release method takes an important next step towards characterization of rare target cells.

<table><tbody><tr><td>Gilupi Release Buffer</td><td>Gilupi</td><td>GIL083</td><td>Used to detach cells from the wire</td></tr><tr><td>McCoy&#039;s 5A Medium (1x) suppl. with L-Glutamine</td><td>Thermo Fisher Scientific</td><td>16600-082</td><td>Culture medium used for HT-29 cells, adapt culture medium to cell line used for the experiments</td></tr><tr><td>HEPES Buffer Solution (1 M)</td><td>PAA</td><td>S11-001</td><td>Supplement for McCoy&#039;s 5A Medium</td></tr><tr><td>Foetal Bovine Serum Gold</td><td>PAA</td><td>A15-151</td><td>Supplement for McCoy&#039;s 5a Modified Medium</td></tr><tr><td>Penicillin-Streptomycin (100x)</td><td>Biowest</td><td>L0018-100</td><td>Supplement for McCoy&#039;s 5a Modified Medium</td></tr><tr><td>Phosphate Buffered Saline (1x PBS)</td><td>Thermo Fisher Scientific</td><td>10010-015</td><td></td></tr><tr><td>Accutase</td><td>Biowest</td><td>L0950-100</td><td>Cell detachment solution (cell culture)</td></tr><tr><td>Water bath</td><td>GFL</td><td>Type 1003</td><td>Used for prewarming solutions</td></tr><tr><td>Incubator</td><td>Thermo Fisher Scientific</td><td></td><td>Used to incubate cells (cell culture)</td></tr><tr><td>50 mL reaction tubes</td><td>VWR</td><td>525-0403</td><td></td></tr><tr><td>Roller mixer</td><td>Stuart Scientific</td><td>SRT6D</td><td>Used to attach cells to the wire while keeping the cells from sedimenting</td></tr><tr><td>CellTrace CFSE Cell Proliferation Kit</td><td>Thermo Fisher Scientific</td><td>C34554</td><td>Used to label living cells (cytoplasmic label)</td></tr><tr><td>Hoechst 33342</td><td>H3570</td><td>Thermo Fisher Scientific</td><td>Used to stain DNA (nucleic counterstain)</td></tr><tr><td>Centrifuge (equipped for holding 15 mL and 50 mL reaction tubes)</td><td>Thermo Fisher Scientific</td><td>Heraeus Megafuge 40R</td><td>Used for pelleting cells</td></tr><tr><td>Observer Z1 (Fluorescence/laser microdissection microscope)</td><td>Carl Zeiss Microimaging</td><td></td><td>Inverted (immunofluorescence) microscope capable of laser microdissection; Fluorescence microscopes must be able to detect Hoechst 33342 (DAPI filter) and CFSE (FITC filter)</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A4503-50G</td><td>Used for coating glass/plastic surfaces</td></tr><tr><td>MS1 Minishaker</td><td>Sigma-Aldrich</td><td>Z404039</td><td>Used for vortexing</td></tr><tr><td>Vacutainer EDTA (or Na-heparin) tubes</td><td>BD</td><td>366450 (or 366480)</td><td>Used as reaction tube for ex vivo capture of target cells</td></tr><tr><td>Detektor CANCER03 DC03</td><td>Gilupi</td><td>GIL003</td><td>Functionalized wire capable of isolating and detaching EpCAM-positive cells (also referred to as catch&amp;amp;release, C&amp;amp;R)</td></tr><tr><td>Syringe needles (G20)</td><td>VWR</td><td>613-0554</td><td>Used to puncture rubber caps in order to allow the non-functional part of the C&amp;amp;R to lead through it</td></tr><tr><td>Neubauer chamber</td><td>Roth</td><td>T729.1</td><td>Used to count cells</td></tr><tr><td>Pasteur pipettes 150 mm</td><td>Volac</td><td>D810</td><td>Used for the low-volume application of cells to the C&amp;amp;R</td></tr><tr><td>Grease pen</td><td>Dako</td><td>S2002</td><td>Used on glass slides to hold cell suspension in place</td></tr><tr><td>Steril syringe filter (0.2 &amp;micro;m)</td><td>Corning</td><td>431219</td><td>For filtering freshly prepared Gilupi Release Buffer</td></tr><tr><td>Delfia PlateShaker</td><td>Perkin Elmer</td><td>1296-003</td><td>Used for agitation during cell detachment</td></tr><tr><td>1.5 mL tubes</td><td>Eppendorf</td><td>30,125,150</td><td></td></tr><tr><td>Ampli1 WGA Kit</td><td>Silicon Biosystems</td><td></td><td>To be ordered via Silicon Biosystems</td></tr><tr><td>Axiovert M200 equipped with Mikromanipulator MMJ and CellTram vario</td><td>Zeiss/Eppendorf</td><td></td><td>Use micromanipulation at hand</td></tr><tr><td>Microcapillaries (25 &amp;micro;m in diameter), CustomTip Type I</td><td>Eppendorf</td><td>930001201</td><td>Used for micromanipulating cells</td></tr><tr><td>0.2 mL PCR tubes</td><td>Biozym</td><td>710920</td><td></td></tr><tr><td>Cytocentrifuge and equippment</td><td>Hettich</td><td>Universal 32</td><td>Use cytocentrifuge at hand</td></tr><tr><td>Thermo cycler</td><td>Bio-Rad</td><td>DNA Engine Dyad</td><td>Every thermo cycler with heated lid can be used</td></tr><tr><td>Microcentrifuge</td><td>Roth</td><td>CX73.1</td><td>Use desk-top centrifuge at hand</td></tr><tr><td>MembraneSlide 1.0 PET</td><td>Zeiss</td><td>415101-4401-050</td><td>Membrane-coated glass slides used for laser microdissection</td></tr><tr><td>UV Stratalinker 1800</td><td>Stratagene</td><td>400072</td><td>Use DNA cross linker at hand</td></tr><tr><td>Standard PCR reagents</td><td></td><td></td><td></td></tr><tr><td>6x DNA Loading</td><td>Thermo Fisher Scientific</td><td>R0611</td><td>Use gel loading dye at hand</td></tr><tr><td>DNA ladder</td><td>BioLabs</td><td>N0551G</td><td>Use 100 bp ladder at hand</td></tr><tr><td>Agarose</td><td>Biozym</td><td>840004</td><td></td></tr><tr><td>Gel electorphoresis station</td><td>Bio-Rad</td><td></td><td>Use electrophoresis system at hand</td></tr><tr><td>Gel Red Nucleic Acid Stain</td><td>Biotium</td><td>41003</td><td>Intercalating dye for DNA visualization In agarose gels</td></tr></tbody></table>

target cell pre-enrichment and whole genome amplification for single cell downstream characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

Cells after CFSE and nucleic staining can be evaluated using an immunofluorescence microscope equipped with filters for DAPI and FITC. Nuclei present with a bright counterstain in the DAPI channel whereas cytoplasm of the cells show uniform labelling with CFSE with heterogeneous inter-cell intensity (Figure 2A and 2E). Similar shape and staining intensities are expected from cells attached to the wire (Figure 2B and 2F) as well as detached from the wire and recovered on a slide (Figure 2D and 2H).
High cell densities (e.g. &#62;1 000 000 cells/mL) may result in total coverage of the wire with cells when charging the wires. However, lower cell densities, changes in rotation speed during incubation, and the use of different cell lines affect the isolation efficiency and need to be tested separately. When the wires were incubated with 5 mL of HT-29 cells at 100 000 cells/ mL as described in section 2.1., a mean of 254 &#177; 103 cells (n = 5) were captured on the wires. With the same experiment using LNCaP cells, a mean of 440 &#177; 319 cells (n = 5) per wire was obtained.
Presenting 500, 5 000, and 50 000 HT-29 cells in a background of 5 mL of peripheral blood to wires (according to protocol section 2.2.) resulted in the capture of 75 &#177; 18 cells, 25 &#177; 9 cells, and 47 &#177; 57 cells (n = 3, each), respectively. If the wire is charged with 15 &#181;L of cell suspension (1 000 000 cells/mL) according to the protocol in section 2.3., up to 1 000 (HT-29) cells may attach to a wire.
Cell detachment efficiencies ranged from 81% in HT-29 cells (range 54.9% - 93.2%, and n = 5) to 81% (range 63.51%-92.87%, n = 6) and 91% (range 84.7% - 95.3%, n = 6) in LNCaP and VCaP cells, respectively. Similarly, the recovery of detached cells differs between cell lines: A mean of 28% of detached HT-29 cells were recovered by cytocentrifugation. Whereas the recovery rate for LNCaP was lower than 10%, the mean recovery rate for VCaP cells was 55%.
Approximately 75% of single cells subjected to WGA yield high quality WGA products: This is 1) smear of WGA products ranging from 0.1 to &#62; 1 kb (Figure 4, top panel) and 2) three or four bands in the 4plex quality control PCR (Figure 4, bottom panel). WGA products can be used for 1) array-CGH profiling meeting the quality criteria for single-cell array-CGH profiles 6,7 and 2) targeted NGS after re-amplification WGA 7.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56394/56394fig1.jpg" /> 
Figure 1: Workflow for isolating and recovering target cells for single-cell analysis. (A) Cells are cultured to 90% confluency and (B) harvested to obtain a single-cell suspension. After staining with CFSE and nucleic stain, (C) the functionalized wire is incubated in the presence of the cells either in a volume of 5 mL using reaction tubes or in a low volume using a Pasteur pipette tip. The latter allows for application of cell suspension volumes as low as 15 &#181;L. (D) Cells need to be detached within 4 h after charging. Therefore, the wire is placed into a 1.5 mL reaction tube filled with release buffer. After 15 min incubation on a rocker and 10 min centrifugation, the wire is removed and the cell suspension is centrifuged to pellet the cells. (E) Recovered cells are either transferred to a glass slide for single-cell micromanipulation (top) or cytocentrifuged onto a membrane-coated slide and forwarded to laser microdissection (bottom). (F) Finally, harvested single cells are amplified by means of whole genome amplification (WGA). WGA products are compatible with array-CGH and targeted NGS downstream analysis.EpCAM epitope: green circle on cell surface. Functionalized wire: Device, grey triple-helical wires. Polymer: purple lines. Anti-EpCAM antibodies: orange. Release buffer activity: red arrow. Grease marker to hold cell suspension in place: dark blue ellipse. Recovered cell suspension: light blue. Capillary for micromanipulation: black line. Magnification: recovered cell harvested by micromanipulation, cytospin on membrane-covered slide containing recovered cells (grey filled ellipse). Magnification: recovered cell being laser microdissected (grey circular line: membrane from membrane slide serving as backbone for laser microdissection), objective with laser beam (blue line approaching the membrane slide from below). <a href="https://cloudflare.jove.com/files/ftp_upload/56394/56394fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56394/56394fig2.jpg" /> 
Figure 2: Visualizing labelled cells. (A,E) Cells before charging: Cells are labelled with CFSE and counterstained with a DNA intercalating dye showing a range of CFSE (green) signal intensity. (B,F) Labelled cells captured on the wire. (C,G) Wire after cell-detachment procedure. (D,H) Cells detached from wire and recovered by cytocentrifugation onto a slide. CFSE: FITC channel (green). DNA stain: DAPI channel (blue). <a href="https://cloudflare.jove.com/files/ftp_upload/56394/56394fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56394/56394fig3.jpg" /> 
Figure 3: Wire and storage compartment. (A) Compartments of the wire. (B) Detail of the functionalized part. <a href="https://cloudflare.jove.com/files/ftp_upload/56394/56394fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56394/56394fig4.jpg" /> 
Figure 4: Quality control of WGA products obtained from single micromanipulated cells. Top panel: WGA products obtained from single cells typically result in DNA smears ranging from 0.2 kb to &#62; 1.0 kb with a peak intensity at around 0.5 kb. Samples number 1, 7 and 13 (indicated with asterisks) show suboptimal or no DNA smears. Bottom panel: WGA products are of sufficient quality if the 4plex PCR yields three or four of four PCR products (at 100, 200, 300, and 400 bp). Samples 1, 7, 10, and 13 show fewer than three bands and would be excluded from further analysis. Lane M contains a 100 bp ladder and asterisks indicate the samples of insufficient WGA product quality. <a href="https://cloudflare.jove.com/files/ftp_upload/56394/56394fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Primer sequence</td>
			<td>NOTE</td>
		</tr>
		<tr>
			<td>5&#8242;-gtt cca ata tga ttc cac cc-3&#8242;</td>
			<td>100 bp forward primer</td>
		</tr>
		<tr>
			<td>5&#8242;-ctc ctg gaa gat ggt gat gg-3&#8242;</td>
			<td>100 bp reverse primer</td>
		</tr>
		<tr>
			<td>5&#8242;-agg tgg agc gag gct agc-3&#8242;</td>
			<td>200 bp forward primer</td>
		</tr>
		<tr>
			<td>5&#8242;-ttt tgc ggt gga aat gtc ct-3&#8242;</td>
			<td>200 bp reverse primer</td>
		</tr>
		<tr>
			<td>5&#8242;-agg tga gac att ctt gct gg-3&#8242;</td>
			<td>300 bp forward primer</td>
		</tr>
		<tr>
			<td>5&#8242;-tcc act aac cag tca gcg tc-3&#8242;</td>
			<td>300 bp reverse primer</td>
		</tr>
		<tr>
			<td>5&#8242;-aca gtc cat gcc atc act gc-3&#8242;</td>
			<td>400 bp forward primer</td>
		</tr>
		<tr>
			<td>5&#8242;-gct tga caa agt ggt cgt tg-3&#8242;</td>
			<td>400 bp reverse primer</td>
		</tr>
	</tbody>
</table>
Table 1: Primer sequences for 4plex quality control PCR

Watch this Scientific Journal Video about Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization at JoVE.com

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

target-cell-pre-enrichment-whole-genome-amplification-for-single-cell

Research

JoVE Journal

Cancer Research

8.8K Views.  Medical University of Graz. Dit protocol is te herstellen en zeldzame doelcellen een mengsel met de doelsoort achtergrond cellen voorbereiden moleculaire genetische karakterisering op het niveau van één cel. DNA kwaliteit is gelijk aan niet-behandelde afzonderlijke cellen en zorgt voor eencellige toepassing (beide screening gebaseerd en gerichte analyse).

Video: Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Genetics

Transcriptome Analysis of Single Cells

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in culture.1,2,3 In the case of the brain, regions such as the hippocampus contain a complex arrangement of different cell types, each with distinct mRNA profiles. The ability to harvest single cells allows for a more in depth investigation into the molecular differences between and within cell populations. We describe a simple and rapid method for harvesting cells for further processing. Pipettes often used in electrophysiology are utilized to isolate (using aspiration) a cell of interest and conveniently deposit it into an Eppendorf tube for further processing with any number of molecular biology techniques. Our protocol can be modified for the harvest of dendrites from cell culture or even individual cells from acute slices.
We also describe the aRNA amplification method as a major downstream application of single cell isolations. This method was developed previously by our lab as an alternative to other gene expression analysis techniques such as reverse-transcription or real-time polymerase chain reaction (PCR).4,5,6,7,8 This technique provides for linear amplification of the polyadenylated RNA beginning with only femtograms of material and resulting in microgram amounts of antisense RNA. The linearly amplified material provides a more accurate estimation than PCR exponential amplification of the relative abundance of components of the transcriptome of the isolated cell. The basic procedure consists of two rounds of amplification. Briefly, a T7 RNA polymerase promoter site is incorporated into double stranded cDNA created from the mRNA transcripts. An overnight in vitro transcription (IVT) reaction is then performed in which T7 RNA polymerase produces many antisense transcripts from the double stranded cDNA. The second round repeats this process but with some technical differences since the starting material is antisense RNA. It is standard to repeat the second round, resulting in three rounds of amplification. Often, the third round in vitro transcription reaction is performed using biotinylated nucleoside triphosphates so that the antisense RNA produced can be hybridized and detected on a microarray.7,8

In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

Neuroscience

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Purification of Low-abundant Cells in the Drosophila Visual System

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small numbers of cells. Utilizing this technology to study development in the Drosophila visual system, which boasts a wealth of cell type-specific genetic tools, provides a powerful approach for addressing the molecular basis of development with precise cellular resolution. For such an approach to be feasible, it is crucial to have the capacity to reliably and efficiently purify cells present at low abundance within the brain. Here, we present a method that allows efficient purification of single cell clones in genetic mosaic experiments. With this protocol, we consistently achieve a high cellular yield after purification using fluorescence activated cell sorting (FACS) (~25% of all labeled cells), and successfully performed transcriptomics analyses on single cell clones generated through mosaic analysis with a repressible cell marker (MARCM). This protocol is ideal for applying transcriptomic and genomic analyses to specific cell types in the visual system, across different stages of development and in the context of different genetic manipulations.

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...

Reusable Single Cell for Iterative Epigenomic Analyses

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a single cell and requiring data from other cells to distinguish signal from experimental background noise in a single cell. This paper describes a method to reuse the same single cell for iterative epigenomic analyses. 
In this experimental method, cellular proteins are first anchored to a polyacrylamide polymer instead of crosslinking them to protein and DNA, alleviating structural bias. This critical step allows repeated experiments with the same single cell. Next, a random primer with a scaffold sequence for proximity ligation is annealed to the genomic DNA, and the genomic sequence is added to the primer by extension using a DNA polymerase. Subsequently, an antibody against an epigenetic marker and control IgG, each labeled with different DNA probes, are bound to the respective targets in the same single cell. 
Proximity ligation is induced between the random primer and the antibody by adding a connector DNA with complementary sequences to the scaffold sequence of the random primer and the antibody-DNA probe. This approach integrates antibody information and nearby genome sequences in a single DNA product of proximity ligation. By enabling repeated experiments with the same single cell, this method allows an increase in data density from a rare cell and statistical analysis using only IgG and antibody data from the same cell. The reusable single cells prepared by this method can be stored for at least a few months and reused later to broaden epigenetic characterization and increase data density. This method provides flexibility to researchers and their projects.

The present protocol describes a single-cell method for iterative epigenomic analyses using a reusable single cell. The reusable single cell allows analyses of multiple epigenetic marks in the same single cell and statistical validation of the results.

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Immunology and Infection

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Bioengineering

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

This protocol describes the general processes and quality control checks necessary for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations. Sequencing parameters, read alignment, and downstream single-cell bioinformatic analysis are also provided.

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying ...

Biology

Two-Step Cell Digestion of Mouse Carotid Artery

Preparation of a Single-Cell Suspension from Mouse Carotid Arteries for Single-Cell Sequencing

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are clogged by plaque. Revealing the cellular composition of the carotid artery at the single-cell level is essential for treating carotid atherosclerosis. However, there is no ready-to-use protocol for the preparation of single-cell suspensions from carotid arteries. To obtain a suitable protocol for the dissociation of normal carotid arteries at the single-cell level with less damage to cells, we designed a two-step digestion method by integrating the digestion process of collagenase/DNase and trypsin. Acridine orange/propidium iodide (AO/PI) dual-fluorescence counting was used to detect cell viability and concentration, and it was found that the single-cell suspension satisfied the requirements for single-cell sequencing, with the viability of cells over 85% and a high cell concentration. After single-cell data processing, a median of ~2500 transcripts per cell were detected in each carotid artery cell. Notably, a variety of cell types of the normal carotid artery, including vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), and macrophages and dendritic cells (M&#966;/DCs), were concurrently detectable. This protocol may be applied to prepare a single-cell suspension of blood vessels from other tissues with appropriate modifications.

Author Spotlight: Vascular Tissue Dissociation and Exploring Single-Cell Subclusters for Targeted Therapy 

Here we describe a two-step cell digestion protocol for preparing a single-cell suspension of mouse carotid arteries.

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are ...

Single Cell Sampling Using Micromanipulation: A Technique to Isolate Single Cells from Cancer Cell Suspension

Source: Chen S. et al. <a href="https://www.jove.com/video/56394" target="_blank">Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization.</a> J. Vis. Exp. (2018)
This video describes the technique for isolating single cells from a pool of target cells to carry out downstream analysis. In this method, a micromanipulator fitted to a microscope is used for high-precision isolation of a single target cell.

Source: Chen S. et al. Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization. J. Vis. Exp. (2018)
This ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

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