Yoyo 1

Cells were plated at equal density on 24-well tissue culture plates and treated as indicated. Cells were treated with monoclonal anti-CLPTM1L for 24 h followed by 72 h of treatment with gemcitabine at the indicated concentrations. Dead cells were fluorescently stained with Yoyo-1 (Life Technologies) and enumerated on an Incucyte FLR live cell imager (Essen Bioscience). Total cell numbers were then enumerated on the imager by staining with Vybrant Dye Cycle Green (Life Technologies) or by permeabilization to Yoyo-1 with 0.01% Triton X-100. A killing index was calculated for each well by dividing the number of dead cells by the number of total cells. The killing indices of triplicate groups were averaged.

λ-DNA (Roche) was stained with YOYO-1 (Invitrogen) at a ratio of 1 dye per 20 base pairs for 48 hours at 4 °C in 0.5× TBE buffer (pH 8). The contour length of unstained λ-DNA (48.5 kbp) is around 16 μm, which increases somewhat after staining^{47 (link)}. T4 DNA ligase (Roche) had a final concentration of 40 units/ml in a 0.5× TBE buffer (pH 8) that was modified as follows. In order to obtain catalytically active ligase, 1 mM ATP (Roche) and 5 mM MgCl₂ were added as T4 DNA ligase cofactors to catalytically active solutions. This solution was incubated for 1 hour at 16 °C to allow full equilibrization of Mg²⁺, ATP, and T4 DNA ligase. After this incubation the free Mg²⁺ concentration was lowered by adding 2 mM EDTA. This is necessary since YOYO-1 is not a stable intercalator at a free Mg²⁺ concentration of 5 mM. Stained DNA was added to the protein yield a final concentration of 5 μg/mL followed by stabilization for 2 hours at 25 °C. Tests using alternative DNA (human genomic DNA from Roche) and an alternative enzyme provider (New England Biolabs) were conducted to exclude possible contamination. In a subset of experiments, a high concentration (0.6 mg/ml) bovine serum albumin (BSA) was added to prevent sticking to the nanochannel walls.

Cells were plated onto glass coverslips and treated for 48 hr with 2% DMSO followed by 48 hr washout or a further 48 hr treatment. Cells were pulsed with 10 μM BrdU in media with or without DMSO treatment for 1 hr, followed by ice-cold methanol fixation. Coverslips were incubated with 2.5 M HCl for 45 min, neutralized with 0.1 M sodium borate and washed 3 x PBS then 3 x PBS+ (PBS, 1% BSA, 0.1% Tween-20). Coverslips were then incubated in anti-BrdU antibody (1:500; Abcam) diluted in PBS+ for 30 min, washed in PBS+ and incubated in secondary anti-rat Cy3 antibody (1:500) diluted in PBS+ for a further 30 min. DNA was counterstained using YOYO-1 (Invitrogen) and coverslips mounted onto slides (90% glycerol, 20 mM Tris, pH 9.2). Images were acquired using an Axioskop2 (Zeiss, Inc.) microscope fitted with a CoolSNAP HQ camera (Photometrics) using MetaMorph Software (Molecular Devices).

For nanofluidics experiments, DNA was stained with YOYO-1 (YOYO, Invitrogen) in a molar ratio of 1:5 to the total number of basepairs in the sample, and with netropsin (Sigma-Aldrich) in a molar ratio of 150:1 with respect to YOYO. The samples were initially mixed in 5x TBE (Tris-Borate-EDTA, Medicago, diluted with ultrapure water from 10x tablets) and left to equilibrate at room temperature for about 20 minutes. As an example, 2 μL of plasmid DNA (100 μM, bp) was mixed with 2 μL λ-DNA (100 μM, bp), 2 μL of YOYO-1 (40 μM) and 3 μL of netropsin (4000 μM). 5x TBE was then added in order to obtain a final volume of 10 μL. Subsequently, the samples were diluted to 0.05x TBE with ultrapure water to a final concentration of typically 0.4 μM of DNA (bp). The mixing in high ionic strength was performed to enable rapid equilibration of YOYO on DNA35 (link). Beta-mercaptoethanol (BME, Sigma-Aldrich) was added in 2% (v/v) to suppress excessive photonicking of the plasmids. λ-DNA (48502 bp, New England Biolabs) was used as standard for measurements of the sizes of the plasmids and was measured in the same conditions as for the plasmids.

After nick-labeling, the samples were treated with Protease and IrysPrep Stop Solution (BioNano Genomics) [32 (link)]. The labeled DNA was stained with YOYO-1 (Invitrogen), and was loaded into the nanochannels following the established protocol [33 (link)]. We generally collected 60x coverage (180Gb) data to obtain 30 molecules containing the telomeres for each chromosome. The image analysis was done following the established procedure [32 (link)].

The DNA samples were treated with Protease and IrysPrep Stop Solution (BioNano Genomics, San Diego, CA, USA) [20 (link)]. The labeled DNA was stained with YOYO-1 (Invitrogen, Carlsbad, CA, USA) before being loaded into the nanochannels following a previously established procedure [24 (link)]. A total of 180 Gb data, which is about 60× coverage, provided around 30 molecules with telomeres for each chromosome arm. The image analysis was performed following the established method [20 (link)].

For optical mapping, the DNA in 4 mm wide agarose fractions was stained with YOYO-1 (Invitrogen) and partially denaturated to produce the DR maps (Supplementary Information). Basic design and working principle of the device used in the experiment is described elsewhere^{11 (link),28 (link)}. Compared to the previous device design^{11 (link),28 (link)}, the present device has been modified so that the slit length is shorter (200 µm) and the cross-shaped slit is narrower at its centre (see Supplementary Fig. S1). Thus shorter molecules (>200 µm) can be stretched and imaged. Stretched DNA molecules were imaged with an Olympus IX81-ZDC2 microscope equipped with 100x/1.40 oil UPlanSApo objective. Images were recorded with an Andor’s iXon3 897 camera using 50-ms exposure and maximum electron multiplying (EM) gain. Molecule images presented in this paper were prepared with ImageJ^{58 (link)} and stitched with the ImageJ plugin Stitching^{59 (link)}. DR maps were aligned with the theoretical prediction for the melting profile of the hg19 human reference genome as previously described in^{11 (link),27 (link),28 (link)}.