Breaks Labeling in situ and sequencing (BLISS)

doi:10.1038/protex.2017.018

Method Article

Breaks Labeling in situ and sequencing (BLISS)

https://doi.org/10.1038/protex.2017.018

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Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity, or practicality. Here, we present Breaks Labeling In Situ and Sequencing (BLISS), featuring: 1) direct labeling of DSBs in fixed cells or tissue sections on a solid surface; 2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; 3) quantification of DSBs through unique molecular identifiers; and 4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells, and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive, and efficient method for genome-wide DSB mapping in many applications.

W.Yan and R.Mirzazadeh equally contributed to this work.

Molecular Biology

Biotechnology

DNA double-strand breaks

BLISS

CRISPR

_In vitro_ transcription

DNA double-strand breaks (DSBs) are major DNA lesions that form in a variety of physiological conditions – such as transcription (1-2), meiosis (3), and VDJ recombination (4) as well as a consequence of exposure to DNA damaging agents and replication stress (5). DSBs can also be induced in a controlled fashion at specific sites in the genome using programmable nucleases, such as the CRISPR (clustered regularly interspaced short palindromic repeats)-associated RNA-guided endonucleases, Cas9 and Cpf1, which have greatly advanced genome editing. However, the potentially mutagenic off-target DNA cleavage activity of these nucleases represents an issue of major concern that needs to be thoroughly assessed before these enzymes can be safely used in the clinical setting (6). Thus, developing methods that can accurately map the genome-wide location of endogenous as well as exogenous DSBs in different systems and conditions is not only essential to advance our understanding of DSB biology, but is also critical for successful translation of programmable nucleases from research tools into clinical applications.

In the past few years, several methods based on next-generation sequencing have been developed to assess DSBs at genomic scale, including ChIP-seq (7-8), BLESS (9-11), GUIDEseq (12), Digenome-seq (13), IDLV-mediated DNA break capture (14), HTGTS (15), and more recently End-Seq (16) and DSBCapture (17). While in general all of these methods represent important complementary tools to detect DSBs genome-wide (Supplementary Table 1), they also have important drawbacks. For example, ChIP-seq of DSB-sensing or repair proteins such as p53 Binding Protein 1 (53BP1) or the phosphorylated variant histone H2A.X (γH2A.X) does not label DSBs directly, and is unable to identify DNA breakpoints with single-nucleotide resolution. GUIDEseq, IDLV-mediated DNA break capture, and HTGTS detect DSBs by quantifying the products of non-homologous end-joining (NHEJ) repair, potentially missing DSBs that are repaired through other pathways. Furthermore, in vivo delivery of exogenous oligonucleotides in GUIDEseq or viral cassettes in IDLV-mediated DNA break capture is challenging especially in primary cells and intact tissues. DSBs induced by programmable nucleases, such as CRISPR-associated RNA-guided Cas9 and Cpf1, can be evaluated in vitro using Digenome-seq, but this approach may not be representative of physiologically relevant conditions – such as chromatin environment and nuclear architecture – that are likely to influence the frequency of DNA breaking and repair, or of relevant nuclease concentrations. Lastly, BLESS and the related methods End-Seq (16) and DSBCapture (17), require substantial amounts of input material (typically, in the order of millions of cells), they are labor-intensive, and are semi-quantitative due to lack of appropriate controls for PCR amplification biases, limiting their applications and scalability. Here, we describe a method for Breaks Labeling In Situ and Sequencing (BLISS)(Figure 1a) that compared to other DSB mapping methods is more versatile, sensitive, and quantitative.

-The list of BLISS adapter used in this protocol is provided in Supplementary Table 2

-Poly-D-Lysine (PDL) (Merck Millipore, cat. no. A003E) (for CRISPR-BLISS)

-Poly-L-Lysine (PLL) solution (Sigma, cat. no. P8920-100 ml) (for BLISS in cells)

-Lipofectamine 2000 (Life Technologies, cat. no. 11668019) (for CRISPR-BLISS)

-OptiMEM (Gibco, cat. no. 31985062) (for CRISPR-BLISS)

-Methanol-free paraformaldehyde (PFA) 16% (EMS, cat. no. 15710)

-Nuclease-free Phosphate-Buffered Saline (10×) pH 7.4 (Thermo, cat. no. AM9625)

-Nuclease-free water (Thermo, cat. no. 4387936) (for in situ reactions and library preparation)

-Lysis buffer 1 (LB1): Tris-HCl 10 mM, NaCl 10 mM, EDTA 1 mM, Triton X-100 0.2%, pH 8 at 4 °C

-Lysis buffer 2 (LB2): Tris-HCl 10 mM, NaCl 150 mM, EDTA 1 mM, SDS 0.3%, pH 8 at 25 °C

-Nucleus Isolation Buffer (NIB): NaCl 146 mM, Tris-HCl 10 mM, CaCl2 1 mM, MgCl2 21 mM, Bovine Serum Albumin 0.05%, Nonidet P-40 0.2%, pH 7.8 (for tissue-BLISS)

-Sucrose for molecular biology (Sigma, cat. no. 57-50-1) (for tissue-BLISS)

-Tissue-Tek® O.C.T. Compound, Sakura® Finetek (VWR, cat. no. 25608-930) (for tissue-BLISS)

-CutSmart® buffer (NEB, cat. no. B7204S)

-Quick Blunting™ Kit (NEB, cat. no. E1201L)

NEBNext® dA-Tailing Module (NEB, cat. no E6053L) (for CRISPR-BLISS)

-T4 DNA Ligase (NEB, cat. no. M0202M)

-UltraPure™ BSA (50 mg/ml) (Thermo, cat. no. AM2616)

-High-salt wash buffer (HSW): Tris-HCl 10 mM, NaCl 2M, EDTA 2 mM, Triton X-100 0.5%, pH 8 at 25 °C

-DNA extraction buffer: SDS 1%, NaCl 100 mM, EDTA 50 mM, Tris-HCl 10 mM, pH 8

-Proteinase K, Molecular Biology Grade (NEB, cat. no. P8107S)

-Nuclease-free TE buffer (Thermo, cat. no. AM9849)

-MEGAscript® T7 Transcription Kit (Thermo, cat. no. AM1334)

-RiboSafe RNase Inhibitor (Bioline, cat. no. 65027)

-DNase I, RNase-free (Thermo, cat. no. AM2222)

-RA3 adaptor and RTP, RP1 and RPI primers (custom-synthesized by Integrated DNA Technologies Inc. based on the sequences in TruSeq Small RNA Library Preparation kit, Illumina)

-RNaseOUT™ Recombinant Ribonuclease Inhibitor (Invitrogen, cat. no. 10777-019)

-T4 RNA ligase 2, truncated (NEB, cat. no. M0242L)

-Deoxynucleotide (dNTP) Solution Set (NEB, cat. no. N0446S)

-SuperScript® III Reverse Transcriptase (Thermo, cat. no. 18080044)

-NEBNext® High-Fidelity 2X PCR Master Mix (NEB, cat. no. M0541L)

-Cell counter (Countess II FL Automated Cell Counter, Thermo)

-Cryostat (Leica Biosystems CM3050 S Research Cryostat) (for tissue-BLISS)

-Incubator (Binder incubator, Model KB 53)

-Tabletop centrifuge (Eppendorf® Microcentrifuge 5424)

-Thermoshaker (Eppendorf® Thermomixer Compact)

-PCR cycler (T3 Thermocycler, Biometra)

-Sonication device (Bioruptor® Plus, Diagenode, cat. no. B01020001)

-DynaMag™-2 Magnet (Thermo, cat. no. 12321D)

-Qubit® 2.0 Fluorometer (Thermo, cat. no. Q32866)

-Bioanalyzer 2100 (Agilent, cat. no. G2943CA)

-SpeedVac (Savant™ SPD131DDA SpeedVac™ Concentrator, Thermo, cat. no. SPD131DDA-220)

Coating of coverslips (for BLISS in cells)

Place the coverslips in a 10 cm dish, cover them with 5 ml PLL solution, and then gently shake the dishes for 15 min Note: make sure that coverslips stay covered by liquid
Aspirate the solution and transfer it into a 15 ml tube Note: the solution can be then used up to three times
Wash the coverslips three times with 1× PBS at room temperature (rt) and once with ethanol (EtOH) 70%
Air-dry the coverslips and proceed with cell spotting and fixation Breakpoint: if not used immediately, coverslips can be stored in EtOH 70% at 4 °C and air-dried just before use.

Attachment and fixation of suspension cells (for BLISS in cells)

Place the desired number of PLL-coated coverslips into a 24-well cell culture plate
In each well, dispense a cell suspension freshly prepared in 1× PBS Note: we usually spot up to 3×10⁵ cells onto a 13 mm coverslip
Let the cells sediment onto the coverslip for 10 min at rt
Slowly add one volume of PFA 8% in 1× PBS equal to the volume of cell suspension added before onto the coverslip
Incubate for 10 min at rt
Rinse the coverslips twice with 1× PBS at rt
Store the samples in 1× PBS at 4 °C or proceed to permeabilization Breakpoint: fixed cells can be stored at 4 °C for several days up to a month. To avoid bacterial contamination, cells should be stored in 1× PBS supplemented with NaN₃ 0.05%.

Transfection and fixation of HEK cells in multi-well plates (for CRISPR-BLISS)

Coat a 24-well cell culture plate with PDL following the same procedure used for coating coverslips with PLL
Seed ~1.25×10⁵ HEK 293T cells per well
Grow cells for 16-18 h until they reach 60–70% confluence
Transfect cells according to your favorite protocol Note: for Cas9-BLISS, we typically use 2 μl of Lipofectamine 2000 and 500 ng of Cas9 plasmid in 100 μl total of OptiMEM per well
After transfection, aspirate the medium and wash the cells in 1× PBS at rt
To each well, add 0.5 ml of PFA 4% in 1× PBS at rt
Incubate for 10 min at rt
Rinse the plate twice with 1× PBS at rt
Store the plate in 1× PBS at 4 °C or proceed to permeabilization

Liver cryopreservation and sectioning (for tissue-BLISS)

Cut the biopsy into small pieces and transfer them into a 15 ml tube containing PFA 4% in 1× PBS
Incubate for 1 h at rt while rotating
Add a volume of glycine 2 M to obtain a 125 mM final concentration
Mix well by pipetting up and down
Wash the tissue fragments twice with 1× PBS at rt
Immerse the tissue fragments in sucrose 15% on a rotator overnight at 4 °C
Immerse the tissue fragments in sucrose 30% on a rotator at 4 °C until they sink at the bottom of the tube
Embed the tissue fragments in OCT and quickly freeze them at –20 °C before cryosectioning
Section the block into 30 μm slices, mounting them onto microscope slides
Air-dry the sections for 60 min at rt
Store the sections at 4 °C or proceed to permeabilization

Preparation of nuclei suspension from liver biopsies (for tissue-BLISS)

Cut the biopsy into small pieces and transfer them into a 2 ml tube containing 1-1.5 ml of NIB buffer
Incubate for 15-40 min at, pipetting the suspension up and down every 5 min, until the tissue fragments become transparent
Centrifuge at 500 g for 5 min at rt
Discard the supernatant
Resuspend the nuclei in 200-500 μl of 1× PBS at rt
Dispense 100-200 μl of the nuclei suspension onto a 13 mm PLL-coated coverslip
Incubate for 10 min at rt
Slowly add one volume of PFA 8% in 1× PBS equal to the volume of nuclei suspension added before onto the coverslip
Incubate for 10 min at rt
Rinse the coverslips twice with 1× PBS at rt
Store the samples in 1× PBS at 4 °C or proceed to permeabilization

DAY 1

Cells and tissue lysis

Briefly rinse the sample in 1× PBS at rt Note: for samples on 13 mm coverslips, this is conveniently done in a 12- or 24-well plate. For tissue-BLISS, the tissue section is covered with a Secure-Seal™ Hybridization Chamber. All the steps until post in situ ligation are done by pipetting 100 μl volumes in and out of the chamber.
Incubate the sample in LB1 for 1h at 4 °C
Briefly rinse the sample in 1× PBS at rt
Exchange to LB2 and incubate for 1 h at 37 °C
Wash the samples and proceed to in situ blunting
Exchange to 1× PBS at rt
Incubate for 1–2 min at rt
Repeat wash in 1× PBS at rt

In situ DSBs blunting

Exchange to 1× CutSmart® buffer at rt
Incubate for 2 min at rt
Repeat twice the equilibration in CutSmart® buffer
Dispense 50 μl of the following blunting mix on piece of Parafilm M® placed in a Petri dish containing a piece of tissue presoaked in distilled water wrapped all around its edge: • Nuclease-free water 37.5 μl

• Blunting buffer 10×* 5 μl

• BSA 10 mg/ml* 0.5 μl

• dNTPs 1 mM* 5 μl

• Blunting enzyme mix* 2 μl

*Components included in Quick Blunting™ Kit

Place the coverslip on top of the blunting mix solution with cells facing downwards
Seal the dish with Parafilm M® Note: the volumes and format are for a 13 mm coverslip. For different sample types, the volumes must be adjusted proportionally. For cells in 24-well plates or tissue sections covered by Secure-Seal™ Hybridization Chamber the blunting mix is added directly into the well or pipetted into the chamber.
Incubate for 1 h at rt

In situ A-tailing (for CRISPR-BLISS)

Exchange to 1× CutSmart® buffer at rt
Incubate for 2 min at rt
Repeat twice the equilibration in CutSmart® buffer
Dispense 50 μl of the following A-tailing mix using the NEBNext dA-Tailing Module on piece of Parafilm M® placed in a Petri dish containing a piece of tissue presoaked in distilled water wrapped all around its edge: ● Nuclease-free water 42 µl

● NEBNext dA-Tailing Buffer (10X)* 5 µl

● Klenow Fragment (3´→ 5´ exo–)* 3 µl

*Components included in NEBNext® dA-Tailing kit

Place the coverslip on top of the A-tailing mix solution with cells facing downwards
Seal the dish with Parafilm M® Note: the volumes and format are for a 13 mm coverslip. For different sample types, the volumes must be adjusted proportionally. For cells in 24-well plates or tissue sections covered by Secure-Seal™ Hybridization Chamber the A-tailing mix is added directly into the well or pipetted into the chamber.
Incubate the samples for 30 min at 37 °C

In situ DSBs ligation

Remove the blunting/A-tailing mix and add 1× CutSmart® buffer at rt Note: for samples on coverslips, this is done by transferring the coverslips from Parafilm M® into a 12- or 24-well plate
Incubate for 2 min at rt
Repeat twice the wash in 1× CutSmart® buffer
Exchange to 1× T4 Ligase buffer
Incubate for 5 min at rt
Dispense 50 μl of the following ligation mix on piece of Parafilm M® placed in a Petri dish containing a piece of tissue presoaked in distilled water and wrapped all around its edge: ● Nuclease-free water 37.5 μl

● T4 ligase buffer 10× 5 μl

● ATP 10 mM 4 μl

● BSA 50 mg/ml 1 μl

● BLISS adapter 10 μM 2 μl

● T4 ligase 0.5 μl

Place the coverslips on top of the ligation mix solution with cells facing downwards
Seal the dish with Parafilm M® Note: the above volumes and format are intended for 13 mm coverslips. For different sample types, the volumes must be adjusted proportionally. For cells in 24-well plates or tissue sections covered by Secure-Seal™ Hybridization Chamber the ligation mix is added directly into the well or pipetted into the chamber
Incubate for 16–18 h at 16 °C

DAY 2

Removal of unligated adapters

Remove the ligation mix and add HSW buffer at rt Note: for samples on coverslips, this is done by transferring the coverslips from Parafilm M® into a 12- or 24-well plate
Incubate 1 h at 37 °C, shaking
Repeat the exchange to fresh HSW buffer and incubation at 37 °C 3–5 times
Exchange to 1× PBS at rt
Incubate for 1–2 min at rt
Exchange to nuclease-free water
Incubate for 1–2 min at rt Note: at this point, samples on coverslips can be air-dried onto a piece of Parafilm M®. For tissue samples, the Secure-Seal™ chamber can be removed from the slide, and the tissue air-dried. For samples in 24-well plates, it is not necessary to dry the cells, as the Proteinase K digestion mix (see below) can be dispensed directly into the well

Extraction of genomic DNA

Dispense 100 μl of the following DNA extraction mix onto the sample: ● DNA extraction buffer 95 μl

● Proteinase K 20 mg/ml 5 μl

Scrape the cells off the coverslips using a sterile cell scraper Note: the above volumes and format are intended for 13 mm coverslips. For tissue sections, the same approach may be used by adjusting the volume depending on the size of the tissue section. Alternatively, the Pinpoint Slide DNA Isolation System™ may be used to capture a portion of the tissue. For cells in 24-well plates, the DNA extraction mix can be dispensed directly into the well after adjusting the volume proportionally
Transfer the solution into a 1.5 ml Eppendorf® RNA/DNA LoBind
Incubate 16–18 h in a thermo-shaker at 55 °C, shaking at 800 rpm

DAY 3

DNA clean-up

Purify the extracted DNA either using silica-based columns or standard phenol-chloroform and isopropanol precipitation protocols
Elute DNA in nuclease-free TE buffer
Measure DNA concentration and then dilute DNA in TE buffer to the concentration recommended for the sonication instrument used (see below)

DNA fragmentation by sonication

Sonicate DNA aiming to achieve a mean fragment size of 300–500 bp. Examples of instruments and settings that we have successfully used are summarized below: • Bioruptor® Plus, for cells with the setting; 30 s ON, 90 s OFF, high mode, 20 cycles in 100 μl TE

• Bioruptor® Plus, for tissue samples with the setting; 30 s ON, 90 s OFF, high mode, 40 cycles in 100 μl TE

• Covaris S-series, for cells and tissue samples with the setting; duty 10%, intensity 4, time 30 s, cycle/burst 200, 4 cycles in 50 μl TE/H2O

DNA concentrating using AMPure beads (option 1)

Pre-warm AMPure XP beads for 30 min at rt
Add a 0.8× ratio of AMPure XP bead suspension to the extracted DNA
Mix thoroughly by pipetting up-down 5-6 times
Incubate for 5 min at rt
Place the sample on a magnetic stand
Incubate for 5 min until all the beads have attached to the magnet
Aspirate and discard the supernatant
With the sample on the magnetic stand, add 200 μl ice-cold EtOH 80%
Aspirate the supernatant
Repeat once the wash in ice-cold EtOH 80%
Air-dry the beads for 5 min at rt
Remove the sample from the magnetic stand
Resuspend the beads in 10 μl of nuclease-free water
Incubate for 5 min at rt
Place the sample on the magnetic stand
Incubate for 5 min until all the beads have attached to the magnet
Transfer 8 μl of the cleared solution into a 0.5 ml Eppendorf® RNA/DNA LoBind tube
Measure DNA concentration using the Qubit® dsDNA HS Assay Kit Breakpoint: at this point, samples can be stored at –20 °C until in vitro transcription

DNA concentrating using SpeedVac (option 2)

Place the extracted DNA samples in the speedVac instrument with the heat temperature set on medium
Centrifuge for 20 min
Keep 8.5 μl of concentrated DNA
Measure DNA concentration using the Qubit® dsDNA HS Assay Kit Breakpoint: at this point, samples can be stored at –20 °C until in vitro transcription

In vitro transcription (IVT)

In a 0.5 ml Eppendorf® RNA/DNA LoBind tube prepare the following mix: ● Sonicated genomic DNA 7.5 μl

● rNTPs mix*^ 8 μl

● T7 polymerase buffer 10×* 2 μl

● T7 polymerase* 2 μl

● RiboSafe RNase Inhibitor 0.5 μl

*Components included in the MEGAscript® kit

^Prepared by mixing equal volumes of each rNTP solution provided with MEGAscript® kit

Incubate for 14 h at 37 °C in a thermocycler with lid set at 70 °C

DAY 4

Removal of genomic DNA

Add 1 μl of DNase I, RNase-free to the IVT product
Incubate 15 min at 37 °C

RNA cleanup

Pre-warm Agencourt RNAClean XP beads for 30 min at rt
Transfer the IVT product into a 1.5 ml Eppendorf® RNA/DNA LoBind tube
Bring the volume up to 30 μl with nuclease-free water
Add a 1.8× ratio of RNAClean XP beads suspension to the IVT product
Incubate for 10 min at rt
Place the sample on a magnetic stand
Incubate for 5 min until all the beads have attached to the magnet
Aspirate and discard the supernatant
With the sample on the magnetic stand, add 200 μl of freshly prepared EtOH 80%
Incubate at least 1–2 min at rt
Aspirate the supernatant
Repeat twice the wash with EtOH 80%
Air-dry the beads for 10 min at rt
Remove the sample from the magnetic stand
Resuspend the beads in 6 μl of nuclease-free water
Incubate for 5 min at rt
Place the sample on the magnetic stand
Incubate for 5 min until all the beads have attached to the magnet
Transfer 6 μl of the cleared solution into a 0.5 ml Eppendorf® RNA/DNA LoBind tube Checkpoint: 1 μl of purified IVT product can be checked on Bioanalyzer 2100 using a RNA 6000 Pico Kit

RA3 Illumina adapter ligation

Note: all the subsequent steps are done on ice, unless otherwise specified

Dilute the RA3 adapter in nuclease-free water 1:5 vol./vol.
Add 1 μl of the diluted RA3 adapter to the sample
In a thermo-cycler, incubate for 2 min at 70 °C with the lid set at 70 °C
Immediately place the sample on ice
Add 4 μl of the following mix: ● T4 RNA Ligase buffer 10× 2 μl

● T4 RNA Ligase 2, truncated 1 μl

● RNaseOUT™ 1 μl

Incubate the sample for 1 h at 28 °C in a thermocycler with the lid set at 30 °C
Transfer the sample on ice
Add 3.5 μl of nuclease-free water

Reverse transcription

Note: all the subsequent steps are done on ice, unless otherwise specified

Prepare a dNTPs mix at 12.5 mM in nuclease-free water
Add 1 μl of RTP primer to the sample
Incubate the sample for 2 min at 70 °C in a thermocycler with the lid set at 70 °C
Immediately place the sample on ice
Add 5.5 μl of the following reverse transcription mix: ● 1^st strand buffer* 2 μl

● dNTPs mix 12.5 mM 0.5 μl

● DTT 100 mM* 1 μl

● SuperScript® III* 1 μl

● RNaseOUT™ 1 μl

*Components included in the SuperScript® III Reverse Transcriptase kit

Incubate the sample for 1 h at 50 °C in a thermocycler with the lid set at 70 °C

Library indexing and amplification

Transfer the sample into a 200 μl PCR tube
Add 2 μl of the desired RPI primer to the sample
Add 35.5 μl of the following PCR mix: ● Nuclease-free water 8.5 μl

● RP1 primer 2 μl

● NEBNext® 2X PCR Mix 25 μl

Perform the following cycles in a thermocycler with the lid set at 105 °C: A. 98 °C, 30 sec

B. 98 °C, 10 sec

C. 60 °C, 30 sec

D. 72 °C, 30 sec

E. Go to B and repeat for 10–16 cycles

F. 72 °C, 10 min

G. 4 °C, hold

Note: the number of PCR cycles should be adjusted empirically based on the amount of genomic DNA used in the IVT reaction. For example, for a 13 mm coverslip fully covered with human diploid cells, we typically use 12–14 cycles

Library clean-up

Pre-warm AMPure XP beads for 30 min at rt
Transfer the PCR product into a 1.5 ml Eppendorf® RNA/DNA LoBind tube
Add a 0.8× ratio of AMPure XP bead suspension to the extracted DNA
Mix thoroughly by pipetting up-down 5-6 times
Incubate for 5 min at rt
Place the sample on a magnetic stand
Incubate for 5 min until all the beads have attached to the magnet
Aspirate and discard the supernatant
With the sample on the magnetic stand, add 200 μl ice-cold EtOH 80%
Aspirate the supernatant
Repeat once the wash in ice-cold EtOH 80%
Air-dry the beads for 5 min at rt
Remove the sample from the magnetic stand
Resuspend the beads in 20 μl of nuclease-free water
Incubate for 5 min at rt
Place the sample on the magnetic stand
Incubate for 5 min until all the beads have attached to the magnet
Transfer 17–18 μl of the cleared solution into a 1.5 ml Eppendorf® RNA/DNA LoBind tube
Measure DNA concentration using the Qubit® dsDNA HS Assay Kit
Store the library at –20 °C Checkpoint: 1 μl of library can be checked on Bioanalyzer 2100 using a High Sensitivity DNA Kit

From the time that samples are ready to be processed, BLISS takes four days.

Proper fragmentation of extracted genomic DNA is crucial for obtaining high quality libraries. Sheared DNA must be checked on the gel or Bioanalyzer 2100 (Agilent Technologies) before proceeding to IVT.

Tagged DSBs by BLISS will generate high quality sequencing libraries with fragment size of 300-500 bp, that can be checked on Bioanalyzer 2100 (Agilent Technologies).

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We would like to thank R. Belliveau for overall research support; R. Macrae for critical reading of the manuscript; and the entire Zhang laboratory for support and advice. We would also like to thank T. Helleday group at Karolinska Institutet for providing reagents and support. W.X.Y. is supported by T32GM007753 from the National Institute of General Medical Sciences and a Paul and Daisy Soros Fellowship. E.W. is a recipient of a Swedish Society for Medical Research (SSMF) Postdoctoral Fellowship. F.Z. is supported by the NIH through NIMH (5DP1-MH100706 and 1R01-MH110049); NSF; the New York Stem Cell Foundation; the Howard Hughes Medical Institute; the Simons, Paul G. Allen Family, and Vallee Foundations; the Skoltech-MIT Next Generation Program; James and Patricia Poitras; Robert Metcalfe; and David Cheng. F.Z. is a New York Stem Cell Foundation-Robertson Investigator. M.B. is supported by the Science for Life Laboratory, the Swedish Research Council (621-2014-5503), and the Ragnar Söderberg Foundation. N.C. is supported by the Karolinska Institutet, the Swedish Research Council (521-2014-2866), the Swedish Cancer Research Foundation (CAN 2015/585), and the Ragnar Söderberg Foundation.

A patent application has been filed including work described in this publication. F.Z. is a cofounder of Editas Medicine and a scientific advisor for Editas Medicine and Horizon Discovery.

supplement0.xlsx
Supplementary Table 2 List of oligo adapters used throughout the paper
supplement0.docx
Supplementary Table 1 Comparison of BLISS with other methods for genome-wide DSB detection

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Breaks Labeling in situ and sequencing (BLISS)

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Anticipated Results

References

Acknowledgements

Additional Declarations

Supplementary Files

Associated Publications

Status:

Version 1

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