Nanoscale photobiotinylation, pulldown and sequencing of region-specific DNA from intact cells

Femto-seq is a novel nanoscale optical method that can be used to obtain DNA sequence information from targeted regions around a specific locus or other nuclear regions of interest. Two-photon excitation is used to photobiotinylate femtoliter volumes of chromatin within the nucleus, allowing for subsequent isolation and sequencing of DNA, and bioinformatic mapping of any nuclear region of interest in a select set of cells from a heterogenous population.


Online methods section.
Photo-biotinylation reagent.Femto-seq utilizes a specifically designed photoactivatable DNA crosslinker with an affinity tag, 4′-Aminomethyltrioxsalen-PEG3-Biotin (AP3B).The photoactivatable component is a psoralen derivative, 4'-aminomethyltrioxsalen (4'-AMT).The compound was synthesized as described in ref 1.The compound has a polyethylene glycol trimer linker to increase solubility.The 3-PEG is terminated with a biotin moiety, which is used as the affinity tag.AP3B is similar to the commercially available EZ-link psoralen-PEG3-biotin probe (ThermoFisher # 29986), but we have found AP3B to be a more efficient photo-crosslinking reagent.

Cell line modifications.
A modified clone of the human bone osteosarcoma epithelial U2OS 2-6-3 cellline 2 was gifted by the Spector Laboratory at Cold Spring Harbor Laboratory.The cells feature ~200 copies of a transgenic construct, each containing 256 Lac Operator (LacO) repeats, 96 tetracyclineresponsive elements which regulate a minimal CMV promoter, and an open reading frame.Transcription of the coding sequences produce CFP-tagged SKL proteins, as well as mRNA containing 24 MS2 sequences when activated.We modified the cell line to stably expresses LacI-YFP to locate the transgene locus by confocal microscopy by accumulation of LacI-YFP at the LacO repeats, and rtTA to enable transcription by the addition of doxycycline.This was accomplished by co-transfection with pSV2-EYFP-LacI plasmid and pCDH puromycin resistant plasmid at a ratio of 1:20.5mM Isopropyl β-D-1thiogalactopyranoside (IPTG, Sigma-Aldrich I5502) was added to the media to allow cell division, since binding of LacI-YFP to the LacO site will block DNA replication.IPTG was replenished every 2-3 days.
1 μg/ml puromycin was added to the select successfully transfected cells.To add the rtTA construct, U2OS 2-6-3 LacI-EYFP cells were co-transfected with pTetOn plasmid and pCDH blasticidin resistant plasmid at a ratio of 1:20.15 μg/ml blasticidin was added to the select successfully transfected cells.
When the distinct single colonies were formed (2-3 weeks after selection started), 72 were selected and plated onto two 48-well plates (with puromycin and IPTG).50 cells survived and were plated on MatTek dishes for imaging.We found 9 that showed CFP-SKL expression after adding doxycycline, and 7 monoclonal cell-lines showed bright yellow spots after IPTG removal.The monoclonal cell-lines that showed bright yellow spots in the nuclei and CFP-SKL expression in the cytosol were frozen for later use.
After allowing the cells to settle on the surface of the plate, 2 mL of DMEM containing 5 mM IPTG and 5 µg/mL doxycycline was added to the dish.Cells were then incubated overnight at 37℃.The following day, 6 hours before irradiation, media was removed and 2 mL of fresh DMEM media without IPTG was added.One hour before irradiation cells were permeabilized by incubation in 0.2% Triton-X-100 in a stabilizing buffer (4% PEG 8000, 1mM EGTA, 10mM PIPES) for 5 minutes.Cells were then washed three times with 1 mL PBS and then incubated in 200 μM AP3B in PBS for 1 hour at 37℃.

Confocal Imaging, ROI targeting and microscope automation.
For the transgene validation experiments reported here, LacI-YFP loci were visualized using a 20x/0.5NAobjective lens and 514 nm excitation.Once a field of view was imaged, a Zeiss Zen VBA macro is run which sends the image data to a dynamic link library (DLL) for analysis.The DLL finds the fluorescent loci and exports a text file of the rectangular ROIs around each locus back into the Zen macro.The loci (fluorescent spots) are found using the algorithm described in ref. 3. The user then uses the photobleaching module to irradiate the ROIs with 700 nm two-photon excitation to photobiotinylate the DNA.The macro and DLL are available from the corresponding author.
In the validation experiment presented here, 4x4 pixel boxes centered on the LacI-YFP locus were used to specify the regions to irradiate.After a field of view was irradiated, the stage was moved to a new field of cells and the process repeated.Around 5000 cells could be irradiated per dish before the YFP contrast was lost (due to LacI-YFP disassociation).In these experiments 30 passes over each ROI was used.
Laser power was ~70 mW delivered through a 0.5 NA objective lens, with a pixel dwell time of 6.3µs and pixel size of 50 x 50 nm.The power we used to activate the AP3B cross-linker and biotinylate the DNA in the transgene experiments was higher than, for example, the powers used in the power series shown in NA and 70 mW, compared to ~70 using a 1.4 NA objective at 12.5 mW (i.e. as in Figure 1E).For our transgene validation experiments, we used higher powers both due to the lower NA and the fact that we Zen and laser irradiation of the regions using 700 nm femtosecond laser pulses is carried out using the ZEN photobleaching module.
do not know the two-photon cross-linking quantum yield.We also were trying to capture a larger volume around the transgene site, so the higher power produced a greater crosslinking depth (i.e. as in Fig. 1E).

Initial optimization experiments.
As we developed the Femto-seq methodology, we carried out a number of initial experiments using the U2OS transgene cells that led to the successful demonstration experiment shown here.These involved different laser powers and ROIs doses, and various "tweaks" to the isolation procedure which we will not cover in detail, with the exception of noting two areas which we have found to be critical.The first is the need for accurate 3D co-alignment of the femtosecond laser focus with the confocal excitation used to locate the target regions.The second is the use of 1,6-hexanediol to help remove non-covalently bound AP3B from the chromatin before streptavidin pulldown.The latter reduced the non-targeted background DNA sequences by more than a factor of 5 (Fig. 2A).The resulting sequencing data is processed using a custom script.Briefly, paired-end reads are mapped to a custom human genome where the transgene plasmid sequences are included as a separate chromosome using bowtie2 software 4 .Mapped reads were visualized using IGV Genome Browser and reads that mapped to regions of interest were counted using bedtools 5 and deeptools software 6 .

Femto-seq Library Preparation Protocol
A. DNA isolation.

B. Biotinylated DNA Pull-down with Streptavidin beads:
1. Thaw fragmented genomic DNA on ice.
2. Resuspend Dynabeads MyOne Streptavidin C1 beads by vortexing.3. Transfer 40 μl of bead slurry per sample to a clean tube.4. Wash the beads 3 times with 5 volumes of TWB buffer (0.05% Tween-20, 5mM Tris-Cl pH 8.0, 0.5 mM EDTA, 1M NaCl).Use magnet to collect beads on the side of the tube and remove the buffer carefully with a pipette. 5. Resuspend beads with 5 volumes of 2X BWB buffer (10mM Tris-Cl pH 8.0, 1mM EDTA, 2M NaCl).6. Add equal volume (200 μl) of pre-washed beads to each sample.7. Incubate at room temperature for 30 min on a rotator (or Thermomixer @600 rpm).8.After binding wash beads, twice with 300 μl of 1x BWB buffer, and twice with 300 μl TWB buffer.9.For each wash, collect the beads on the side of the tube using a magnet for 2 minutes, remove and discard the buffer by pipetting.Transfer beads with the new wash buffer to a new tube each time.

Femto-seq on cells fixed using DNA-FISH protocols.
Metaphase DNA-FISH.DNA-FISH was performed using commercially available FISH PAINT probes from KromaTiD (Longmont, CO) and performed using their standardized protocol.In short, cells were arrested in metaphase with 10 μl of 10 μg/ml colchicine and cells harvested after 4 hrs.After swelling using 75 mM KCl hypotonic solution, suspensions were fixed in 3:1 methanol and acetic acid.20-30 μl of cell suspension was dropped onto glass slides and allowed to dry as per standard metaphase slide preparation 7 .Widefield images were taken using a 63x oil 1.4 NA objective on an inverted Nikon eclipse TI fluorescence microscope.

DNA-FISH Streptavidin
Staining.Alexa-647-Streptavidin staining was performed on Femto-seq photobiotinylated samples to demonstrate the method also works in fixed samples.Samples were both fixed using a 4%PF 3D FISH protocol and 3:1 methanol acetic acid fixation for metaphase preparations.
Samples prepared for metaphase FISH were prepared following the manufacturer's protocol (KromaTiD) as described above.Samples prepared for 3D FISH were prepared following standard protocols 8 .Both types of fixed samples were washed in PBS, then incubated in 200 μM AP3B in PBS for 1 hour at 37°C and immediately used for site specific photobiotinylation.Following photobiotinylation, cells were washed with PBS for 1 hr at 37°C.Then 1 ml of 1:500 dilution of 1 mg/ml Alexa-647-streptavidin was incubated with the cells for 30 minutes.Cells were then washed with PBS for 30 minutes and stained with DAPI mounting solution and imaged.

Figure 1E .
Figure 1E.Adjusting for the differing numerical apertures (1.4 in Figure 1E vs 0.5 in our transgene

Figure S1 .
Figure S1.Use of Zeiss LSM 880 confocal/multiphoton system for Femto-seq.A VBA (Visual Basic for Applications) macro written for the Zeiss Zen software (version 2.3 SP1) sends the image data (2D array of bytes -typically 512 x 512) to a dynamic link library (Femtoseq.dll)written in C/C++ (Visual Studio 2019) which locates the fluorescent loci and generates ROIs.The ROIs are imported back into Zen and laser irradiation of the regions using 700 nm femtosecond laser pulses is carried out using the ZEN photobleaching module.
Following irradiation, cells were washed with 1,6-hexanediol (50mM) for 1 hour on the dish.The cells were then detached from the dish with 0.05% Trypsin-EDTA (Gibco) and spun down and stored at -80℃ until DNA extraction.Genomic DNA from cross-linked (2P or UV) and non-cross-linked cells were extracted and fragmented to 200-800 bp by sonication.Biotinylated fragments are then captured by Dynabeads MyOne C1 streptavidin beads, and while on beads, end repair, A-tailing, and TRUseq adapter ligation was performed.Adapter ligated fragments are then eluted, and psoralen cross-links chemically reversed in the presence of 3M Urea, 0.1 M KOH, 1 mM EDTA at 90℃.The resulting library is PCR amplified and submitted for paired-end Illumina sequencing (2x 37bp) on a HiSeq 2000 or a NextSeq 500 instrument.

Figure S2 .
Figure S2.Representative Metaphase Spreads.A-E Metaphase DNA-FISH on U2OS cells.LacO locus in yellow, Chr1 in cyan, Chr18 in magenta, DAPI in blue.Boxes around the selected chromosomes show the chr1: chr18 translocation and new location of the YFP locus, F Enlarged view of the selected chromosomes showing the translocation of the YFP locus to Chr18.G-K Metaphase DNA-FISH on U2OS cells.LacO locus in yellow, Chr1 in cyan, Chr18 in magenta, DAPI in blue.Boxes around the selected chromosomes show the chr1: chr18 translocation and new location of the YFP locus.L Enlarged view of the selected chromosomes showing the translocation of the YFP to Chr18

Figure S3 .
Figure S3.Demonstration of AP3B photobiotinylation in fixed cells.A. DNA-FISH to the YFP locus in cells fixed in Carnoy's fixative (3:1 methanol and acetic acid) for metaphase spread, white boxes show the areas for localized biotinylation using the 700 nm two-photon targeting.B. Alexa-647-Streptavidin (Magenta) and DAPI (Blue) labeling after photobiotinylation showing that twophoton mediated AP3B biotinylation can be used to in both cells and metaphase chromosomes in Carnoy's fixed cells.C. Representative photobiotinylation in 4% Paraformaldehyde fixed cells prepared for 3D-FISH protocol using a standard 3D-FISH protocol 4 .D. DNA recovery amounts for both non-fixed cells and fixed in 4% PFA cells under untargeted UV box photo-biotinylation.PFA-Fixed 1 and Non-Fixed used the standard ethanol precipitation for DNA purification after crosslink reversal, while PFA-Fixed 2 used a DNA purification column (Zymo Research DNA Clean and Concentrator).
Add 2 μl of 0.3 uM Indexed TRUseq adapter + 3 μl of T4 DNA Ligase (NEB, 1200 units).9. Incubate ligation reaction at 16℃ overnight.10.Next day, wash beads 2x 0.4ml 1x TWB buffer and once with 0.4 ml BWB buffer.Transfer beads to a clean tube with each wash.11.Elute DNA from beads with 56 μl Elution Buffer (10 mM EDTA, 95% Formamide).Incubate at 65℃ for 10 minutes.12. Collect beads on magnet and transfer supernatant to a new tube.13.Collect residual beads on magnet and transfer supernatant to a new tube again.This step is critical -if any residual magnetic beads are left in the elution it will cause DNA degradation during the next crosslink reversal step.Run 12 μl (half) of each PCR reaction on 1.2% Agarose gel and determine the appropriate PCR cycle for the final library amplification.Wash bead pellet 2x with 200 μl 75% EtOH without disturbing the pellet.Discard the wash 19.Air dry beads ~5min at RT. 20.Elute DNA with 20 μl of 10 mM Tris-Cl pH 8.5 (Qiagen EB buffer).21.Quantitate DNA concentration using 2 μl samples with Qubit HS dsDNA Assay.22. Submit samples for Illumina sequencing (ideally paired-end).