Materials and Reagents

1.5 mL Eppendorf tube
96-well microplate, flat bottom, clear (Greiner Bio-One, catalog number: 655101)
Multiplate^TM 96-well PCR plates, low profile, clear (Bio-Rad, catalog number: MLL9601)
Cell line(s) of interest and appropriate media/culture reagents
ALT-positive cell line for source of C-circle positive control/standard curve DNA [e.g., U2OS (ATCC, catalog number: HTB-96), Saos-2 (ATCC, catalog number: HTB-85)]
Telomerase-positive cell line for source of C-circle negative control DNA [e.g., HeLa (ATCC, catalog number: CCL-2), HCT116 (ATCC, catalog number: CCL-247)]
Tris base (Fisher Scientific, catalog number: BP152)
Potassium chloride (KCl) (Fisher Scientific, catalog number: BP366)
Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Fisher Scientific, catalog number: M35)
NP-40 (also known as IGEPAL^® CA-630) (Sigma-Aldrich, catalog number: I3021)
Tween 20 (also known as Polysorbate 20) (Fisher Scientific, catalog number: BP337)
Deionized, nuclease-free H₂O [purified using Q-Gard^® 2 purification cartridge (Sigma-Aldrich, catalog number: QGARD00D2)]
Protease (7.5 AU) (QIAGEN, catalog number: 19155)
QuantiFluor^® ONE dsDNA system (Promega, catalog number: E4871)
Φ29 DNA polymerase [includes 10× Φ29 reaction buffer and 20 mg/mL bovine serum albumin (BSA); New England Biolabs, catalog number: M0269)
dNTP set (100 mM each dNTP) (Thermo Fisher Scientific, catalog number: R0181)
UltraPure^TM dithiothreitol (Invitrogen, catalog number: 15508013)
iQ^TM SYBR^® Green Supermix (Bio-Rad, catalog number: 1708880)
C-circle primers (human and mouse)
Forward: 5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′
Reverse: 5′-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′
36B4 primers (human)
Forward: 5′-CAGCAAGT GGGAAGGTGTAATCC-3′
Reverse: 5′-CCATTCTATCATCAACGGGTACAA-3′
36B4 primers (mouse)
Forward: 5′-ACTAATCCCGCCAAAGCAACC-3′
Reverse: 5′-GTAGCGGTTTTGCTTTTTCATCCT-3′
Quick C-Circle Preparation (QCP) lysis buffer (1×) (see Recipes)
1 M Tris-HCl, pH 8.5 (see Recipes)
Φ29 DNA polymerase master mix (2.16×) (see Recipes)

Equipment

Plate reader (Promega GloMax^® Explorer Multimode Microplate Reader; catalog number: GM3500)
Thermal cycler for RCA (MJ Mini^TM Personal Thermal Cycle; Bio-Rad, catalog number: PTC1148)
Thermal cycler for qPCR [Bio-Rad C1000 Touch^TM Thermal Cycler Chassis (Bio-Rad, catalog number: 1841100) equipped with CFX96 Optical Reaction Module for Real-Time PCR (Bio-Rad, catalog number: 1845097)]

Software

CFX Maestro Software for CFX Real-Time PCR Instruments (Bio-Rad; https://www.bio-rad.com/en-us/product/cfx-maestro-software-for-cfx-real-time-pcr-instruments?ID=OKZP7E15)
Microsoft Excel 2019 (v16.0)

Procedure

The following protocol is optimized for 2 × 10⁵ cells; however, we have successfully used this protocol to isolate and quantify C-circles with up to 5 × 10⁵ cells. If adjusting cell numbers, all volumes should be scaled appropriately. We have successfully quantified C-circle abundance in ALT-positive human (Robinson et al., 2021) and mouse (Robinson et al., 2020) cells isolated from distinct 2D and 3D culture conditions, and from cells subjected to an array of genetic and pharmacologic manipulations. Thus, in our hands, this represents a generalizable protocol for C-circle quantitation across diverse in vitro conditions. Therefore, specific details regarding cell lines and culture conditions are not included here, except where noted as experimental controls.

Note: Established ALT- and telomerase-positive cell lines (see Materials and Reagents) should be cultured in parallel with cell line(s) of interest. These serve as a source of DNA for standard curve generation (ALT) as well as positive (ALT) and negative (telomerase) controls for RCA and C-circle quantification. In our experience, results are best when all DNA (including controls) is isolated simultaneously. Doing so prevents degradation of DNA during storage or repeated freeze-thaw cycling of samples that are shared between experiments (e.g., positive and negative controls).

DNA isolation from cultured cells using Quick C-circle Preparation (QCP)
1. Pre-warm 1× QCP lysis buffer (see Recipe 1) in a 1.5 mL Eppendorf tube in a heat block or water bath set to 56 °C. Best results are achieved when QCP lysis buffer is made fresh for each experiment. Volume of QCP lysis buffer needed = 50 μL × (number of samples + 2) (included excess to account for pipetting error or evaporative loss).
2. If lyophilized, resuspend protease (7.5 AU) in 7 mL of deionized, nuclease-free H₂O. Mix thoroughly and place on ice until use. If previously resuspended, remove from -20 °C storage and thaw slowly on ice.
  Note: QIAGEN protease is preferred to other proteases because it can be heat-inactivated at 65 °C.
3. Liberate cells from culture plates (e.g., trypsinization, nonenzymatic digestion from 3D culture) or remove cells from frozen storage and place on ice. At this stage, samples should be cell pellets rather than suspensions.
4. Add protease to pre-warmed QCP lysis buffer at a ratio of 1:20 (i.e., 2.5 μL of protease to 50 μL of QCP lysis buffer). Mix thoroughly by vortexing and collect by microcentrifugation. This should be done immediately prior to cell resuspension.
5. Resuspend each cell pellet in 50 μL of QCP lysis buffer supplemented with protease. Mix by vortexing at 2,000 rpm for 15 s. Suspensions are viscous and may require additional mixing by pipetting up and down. Be careful not to introduce bubbles during resuspension.
  Note: C-circles mixed with genomic DNA (i.e., in whole-cell lysates) are somewhat protected from shear forces compared to pure circular DNAs. While mixing samples by vortexing is acceptable and used throughout this protocol, repeated vortexing results in small but appreciable C-circle loss (<1% per 15 s vortex). If yield appears to be low during quantitation, vortex speed or frequency may be adjusted to minimize C-circle loss.
6. Incubate cell suspensions at 56 °C for 1 h on a heat block. Samples should be mixed by vortexing for 15 s every 15–20 min.
7. Increase temperature of heat block to 70 °C and incubate for 20 min to inactivate protease. Samples do not need to be mixed during this step.
8. Allow samples to cool slowly to room temperature on a benchtop. Once cooled, mix by vortexing at 2,000 rpm for 15 s and collect any evaporated liquid by microcentrifugation.
9. Samples may be used immediately for DNA quantitation and RCA. Otherwise, flash-freeze on dry ice and store at -80 °C.

Quantitation of total cellular DNA
1. Perform serial dilutions of QuantiFluor^® ONE Lambda DNA (400 ng/μL) (from QuantiFluor^® ONE dsDNA system) to generate dsDNA standards according to the manufacturer’s instructions. Final DNA concentrations are: 400, 200, 50, 12.5, 3.1, 0.8, and 0.2 ng/μL.
2. In a 96-well microplate, transfer 200 μL of QuantiFluor^® ONE dsDNA dye to each well that will contain standards, experimental samples, or blanks (i.e., background fluorescence). Each standard, sample, and blank should be analyzed in duplicate.
3. Using a P2 pipettor, transfer 1 μL of each experimental sample or dsDNA standard prepared in Step B1 to the corresponding wells. For blank wells, add 1 μL of 1× TE buffer.
4. Mix the plate thoroughly using a plate shaker or by pipetting the contents of each well using a multichannel pipette. If using a pipette, do not pipette up the entire volume of each well, and eject slowly to avoid introducing air bubbles, which can interfere with fluorescence detection.
5. Incubate plate for 5 min at room temperature, protected from light.
6. Measure fluorescence using a plate reader. If using the GloMax^® system, select the preloaded protocol “QuantiFluor ONE dsDNA System.” Otherwise, use the following excitation and emission parameters: 504 and 531 nm, respectively.
7. To calculate the DNA concentration of each experimental sample, first subtract the blank fluorescence from that of each standard and sample. Use these corrected values for the dsDNA standards to generate a standard curve of fluorescence intensity vs. DNA concentration (i.e., DNA concentration on x-axis and fluorescence on y-axis). Determine the DNA concentration of each sample using the standard curve equation (Figure 1).
  
  Figure 1. Representative standard curve of QuantiFluor^® ONE Lambda DNA. Standard curve equation (top left) is used to calculate DNA abundance in experimental samples using measured absorbance. Each DNA concentration was analyzed and graphed in duplicate.

Rolling circle amplification (RCA) of extrachromosomal DNA
1. Make up Φ29 DNA polymerase master mix (see Recipe 3) and store aliquots at -20 °C if not used immediately. We have used frozen master mix up to one month after initial freezing with no appreciable impact on RCA efficiency.
2. If thawing QCP samples, place on ice to thaw slowly. Once thawed, mix by vortexing at 2,000 rpm for 15 s and collect by microcentrifugation. Keep samples and master mix on ice while setting up RCA reactions.
3. For samples isolated using QCP (including ALT-positive and ALT-negative controls), add 1 μL to 9 μL of 10 mM Tris, pH 7.6 in two 0.2 mL PCR tubes. Depending on the concentration of each sample, they may be diluted in 10 mM Tris, pH 7.6 to avoid pipetting small volumes. However, the total quantity of QCP buffer in each reaction must be the same and should not exceed 1 μL.
4. Aliquot 9.25 μL of Φ29 master mix for each reaction (plus excess, i.e., enough for two extra reactions) into an Eppendorf tube.
5. Remove Φ29 DNA polymerase from -20 °C storage and keep on ice or in a portable cooling block. Add 0.75 μL of polymerase per reaction (plus excess) to tube containing master mix to generate the final reaction mix. Mix by gently flicking the tubes and place on ice.
  Note: Excess polymerase is used relative to template to ensure linear amplification of circular DNAs (rather than logarithmic, which could confound comparative analysis). Other polymerases (e.g., Klenow fragment of DNA polymerase I) may be used if they possess processivity and strand displacement capability as Φ29 polymerase.
6. Repeat step C4, aliquoting into a new Eppendorf tube. Add 0.75 μL of Milli-Q H₂O per reaction to this tube. This serves as the final reaction mix for Φ29-deficient reactions, which are performed for each sample.
  Note: Sample-specific background can be corrected by running a Φ29-deficient RCA reaction in parallel for each sample. This is important for C-circle quantitation by qPCR because denaturation during PCR enables the detection of linear telomeric DNA. In addition, the inclusion of Φ29-deficient controls is particularly useful for samples with low or unknown ALT activity, as these may exhibit relatively high background signal.
7. Add 10 μL of final reaction mix (Φ29-containing and Φ29-deficient) to one tube containing each QCP sample. Mix by gently vortexing (<1,500 rpm) for 5 s.
8. Incubate tubes in a thermal cycler according to the following thermal profile:
  1. 30 °C for 4–8 h (RCA).
    Note: Increasing amplification time increases sensitivity at the expense of reduced linearity. If absolute C-circle abundance is known or expected to be high, 4 h is sufficient for amplification. If C-circle abundance is low or unknown, incubation should be increased to 8 h to maximize sensitivity. Reactions should not be extended beyond 8 h, as amplification becomes nonlinear at that time.
  2. 70 °C for 20 min (Φ29 inactivation).
  3. 4 °C for ∞.
  Typically, we perform RCA for 8 h and allow to run overnight. Samples are stable at 4 °C until convenient to collect the following morning.
9. Generally, we use RCA reactions immediately for C-circle quantitation. However, reactions may also be stored at -20 °C for up to two weeks or at -80 °C for up to one month without appreciable DNA loss.

Determination of C-circle abundance using qPCR
1. Thaw SYBR^® Green Supermix and primers from -20 °C storage on ice. Protect SYBR^® Green Supermix from light.
2. Each sample will be used to quantify C-circle abundance as well as the abundance of the single-copy gene ribosomal protein lateral stalk subunit P0 (also known as 36B4; see Materials and Reagents). Single-copy gene abundance provides a measure of total DNA content, which can be used to normalize C-circle abundance and enable comparative analysis (see Data Analysis). Thus, all subsequent steps should account for the fact that both targets will be measured (e.g., making sufficient master mix).
3. Dilute DNA isolated from an ALT-positive cell line (e.g., U2OS, Saos-2) by QCP to obtain samples for generating a standard curve. For example, for a starting DNA concentration of 3.2 ng/μL, perform five two-fold serial dilutions for a final dilution series containing 3.2, 1.6, 0.8, 0.4, 0.2, and 0.1 ng/μL DNA. To account for differences in PCR efficiency between telomeric DNA and 36B4, each sample will be analyzed in duplicate for each target. Keep diluted samples on ice until ready to load PCR plate.
  Note: RCA is not required prior to dilution and standard curve generation.
4. Assemble qPCR master mixes according to the recipe outlined in Table 1. In addition to Φ29-containing and Φ29-deficient RCA reactions, include a no-template control (using H₂O in place of template DNA) to account for DNA contamination of PCR reagents. The C_t values for this no-template control should be at least four cycles greater than the C_t value of the lowest concentration on the standard curve.
  
  Table 1. Recipe for qPCR master mix.
  Reagent Final concentration Volume (μL)
  2× SYBR^® Green Supermix 1× 12.5
  Forward primer (10 mM) 500 nM 1.25
  Reverse primer (10 mM) 500 nM 1.25
  RNase-free H₂O - 6
5. Dilute each RCA sample to 0.5 ng/μL in 10 mM Tris, pH 7.6, using the concentrations calculated in Section B.
  Note: qPCR is inhibited by both Φ29 polymerase and the inorganic pyrophosphate byproduct of RCA. To avoid qPCR inhibition, limit the RCA reaction that is added to each qPCR reaction or pretreat each RCA sample with a pyrophosphatase. Given the sensitivity of qPCR, it is generally best to limit the quantity of undiluted RCA reaction that is added to <5% of the qPCR reaction volume.
6. Add 4 μL (i.e., 2 ng) of template DNA (Φ29-containing or Φ29-deficient) or RNase-free H₂O (for no-template control) to pre-designated wells of a 96-well PCR plate. Each sample should be analyzed in triplicate.
7. Add 21 μL of qPCR master mix to each well containing DNA or H₂O. Mix by gently vortexing the plate for 5 s and collect volume by centrifugation.
8. Incubate plate in a real-time thermal cycler according to the following thermal profile:
  1. 95 °C for 15 min
  2. 95 °C for 15 s
  3. 54 °C for 2 min
  4. 72 °C for 1 min
  5. Repeat steps b–d 34 times

Data analysis

To construct the telomeric DNA and single-copy gene standard curves, plot C_t (y-axis) vs. DNA concentration (x-axis; log scale) for each duplicate of the six standard DNA concentrations in Microsoft Excel. Draw a standard curve by fitting a best-fit line to the data (Figure 2). There should be good linearity of the standard curve (R² > 0.98, p < 0.05).
In addition, the CFX Maestro software can be used to draw a standard curve to calculate qPCR efficiency, which describes how much of a target amplicon is being produced during each PCR cycle. To construct a standard curve in the CFX Maestro software, change designation of wells containing standard samples (i.e., from ALT-positive cell line; see step D3) to “Standard” on the plate setup (Settings → Plate Setup → View/Edit Plate). In addition, manually enter the DNA concentration in each standard well. The standard curve, along with the calculated efficiency, will now appear under the “Quantification” tab. For C-circle abundance (CCA) quantitation, efficiency should be close to 100% (± 5%).
Note: Examine all C_t values for the single-copy gene to identify any abnormally high or low values, which could indicate unexpected copy number loss or gain, respectively. Discrepancies secondary to copy number alteration may complicate quantitative assessment of CCA and the assignment of ALT status and necessitate repeating the qPCR with another single-copy gene.

Figure 2. Representative standard curve of Saos-2 DNA. C_t values were obtained using telomere-specific qPCR primers. Standard curve equation (bottom left) was used to calculate DNA abundance in experimental samples using sample-specific C_t values and was repeated for single-copy gene. Each DNA concentration was analyzed and graphed in duplicate.
Determine the relative telomeric content (TC) and relative single-copy content (SC) for each replicate using the appropriate standard curve equation, using the C_t values obtained by qPCR. This calculation will provide the total TC (i.e., chromosomal and extrachromosomal) rather than specifically C-circle content.
Calculate the mean (μ) TC and SC and standard deviation (σ) for each sample. Use these values to calculate the coefficient of variation (CV) for each sample according to the following:

CVs for TC (Φ29-containing) should be <15%, while CVs for TC (Φ29-deficient) and SC should be <10%.
Calculate normalized telomere content (NTC) by dividing the average TC by the average SC from the same sample. The CV for NTC is estimated as the sum of the CVs for TC and its matched SC for a given sample.
Calculate CCA in each sample according to the following:
CCA=NTC_Φ29-NTC_{No Φ29}
Calculate the mean NTC for each experimental condition. Construct error bars for CCA using these mean values and the NTC CVs calculated in step 4, as follows:
Upper error=(μ_Φ29+CV_Φ29)-(μ_{No Φ29}-CV_{No Φ29})= (μ_Φ29-μ_{No Φ29})+(CV_Φ29+CV_{No Φ29})
Lower error=(μ_Φ29-CV_Φ29)-(μ_{No Φ29}+CV_{No Φ29})= (μ_Φ29-μ_{No Φ29})-(CV_Φ29+CV_{No Φ29})
Plot CCA mean (step 5) and error (step 6) for each condition on a bar graph. If comparing groups, use the appropriate statistical test for comparison of means (i.e., Student’s t-test for two means, ANOVA for more than two means, or nonparametric equivalents).
Assign TMM identity to each experimental group. A group is considered ALT-positive if (i) its CCA mean is >1% of the ALT-positive control; and (ii) its CCA lower error is nonnegative.

Recipes

Quick C-Circle Preparation (QCP) lysis buffer (1×)

Stock reagent Final concentration Volume (per 100 mL)
1 M Tris-HCl, pH 8.5 10 mM 1 mL
5 M KCl 50 mM 1 mL
1 M MgCl₂ 2 mM 200 μL
NP-40 0.5% (v/v) 500 μL
Tween 20 0.5% (v/v) 500 μL
Milli-Q H₂O - 96.8 mL

1 M Tris-HCl, pH 8.5
For 100 mL, dissolve 12.11 g of Tris base in 80 mL of Milli-Q H₂O.
Adjust pH to 8.5 by adding concentrated HCl dropwise with continuous stirring.
Bring volume to 100 mL with Milli-Q H₂O.

Φ29 DNA polymerase master mix (2.16×)

Stock reagent	Final concentration	Volume (per 925 μL)
10× Φ29 reaction buffer	2.16×	200 μL
20 mg/mL BSA	432 μg/mL	20 μL
Tween 20 (10% v/v in H₂O)	0.216% (v/v)	20 μL
100 mM dATP	2.16 mM	20 μL
100 mM dGTP	2.16 mM	20 μL
100 mM dTTP	2.16 mM	20 μL
100 mM dCTP	2.16 mM	20 μL
1 M dithiothreitol	8.65 mM	8 μL
Milli-Q H₂O	-	597 μL

Acknowledgments

We are grateful to all authors (M. Miyagi, J.A. Scarborough, J.G. Scott, and D.J. Taylor) of the original research paper from which this protocol is derived (Robinson et al., 2021) for their contributions to that work, including the design and implementation of this protocol. We also thank members of the Schiemann laboratory for their assistance in crafting and critically appraising this manuscript. Funding support was provided by the National Institutes of Health to W.P.S. (CA236273) and N.J.R. (F30 CA213892). Additional support was graciously provided to W.P.S. by the Case Comprehensive Cancer Center’s Research Innovation Fund, which is supported by the Case Council and Friends of the Case Comprehensive Cancer Center.

Competing interests

The authors declare that they have no compe ting interests.

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