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| # | Action | Timing |
|---|---|---|
| 1 | Extract genomic DNA from ~5-10×10^6 edited living cells (HEK293T cells) using CWBIO universal gDNA kit following the manufacturer's instructions | 20-40 min |
| 2 | Quantify DNA using NanoDrop spectrophotometer | |
| 3 | Check editing efficiency at on-target sites by Sanger sequencing or targeted amplicon sequencing | |
| 4 | Load 3.75 μg gDNA into Covaris microTUBE supplemented with 10 mM Tris-HCl (pH 8.0) to total volume of 130 μl | |
| 5 | Shear gDNA to average size of 300 bp using Covaris ME220 settings: duration 70 s, peak power 70 W, duty factor 20%, cycles per burst 1000, average power 14 | 70 s |
| 6 | Fragment another 3.75 μg of quantified gDNA for each sample to obtain ≥5 μg sheared DNA after purification | |
| 7 | Purify fragmented DNA with Vistech DNA Clean & Concentrator 5 kit using 5 volumes DNA binding buffer per volume of DNA sample | |
| 8 | Elute with 60 μl nuclease-free water at room temperature | |
| 9 | Quantify DNA using NanoDrop spectrophotometer | |
| 10 | Assemble end-repair reaction: DNA sample (~5 μg), spike-in model sequences (2.5-10 pg each), 10 μl end repair reaction buffer, 5 μl end repair enzyme mix, 2 μl 50 mM NAD+, 2 μl E. coli DNA ligase, nuclease-free water up to 100 μl total | |
| 11 | Pipette on ice to mix thoroughly | |
| 12 | Incubate in thermocycler at 20°C for 30 min | 30 min |
| 13 | Purify DNA with AMPure XP beads using ratio of 2× beads to sample (e.g., add 200 μl to 100 μl sample) | |
| 14 | Mix by briefly vortexing and incubate at RT for 10-15 min | 10-15 min |
| 15 | Place tubes on magnetic plate until liquid is clear, then discard supernatant | |
| 16 | Wash beads with 80% (vol/vol) ethanol, 500 μl per wash, incubate 30 s, aspirate and discard | |
| 17 | Repeat 80% ethanol wash and air-dry pellet for 5-10 min | 5-10 min |
| 18 | Resuspend pellet in 40 μl nuclease-free water by pipetting up and down thoroughly | |
| 19 | Incubate at RT for 5-15 min, then transfer supernatant to new PCR tube | |
| 20 | Mix 40 μl DNA sample with 40 μl of 2× EtONH2 buffer | |
| 21 | Incubate at 37°C for 6 h in thermomixer with shaking at 850 rpm | 6 h |
| 22 | Dilute reaction by adding 120 μl nuclease-free water | |
| 23 | Purify DNA with Vistech DNA Clean & Concentrator 5 kit | |
| 24 | Elute with 42 μl nuclease-free water at RT | |
| 25 | Set up dA-tailing reaction: 42 μl DNA sample, 5 μl dA-tailing reaction buffer, 3 μl Klenow fragment (3'-5' exo-) | |
| 26 | Pipette to mix and incubate in thermocycler at 37°C for 30 min | 30 min |
| 27 | Purify DNA with AMPure XP beads (2× ratio), elute in 38 μl nuclease-free water | |
| 28 | Prepare damage repair reaction: 38 μl DNA sample, 5 μl NEBuffer 3.0 (10×), 1 μl 50 mM NAD+, 1 μl 2.5 mM dNTPs, 2 μl Endo IV, 1 μl Bst full-length polymerase, 2 μl Taq DNA ligase | |
| 29 | Pipette to mix and perform reaction in thermocycler: 37°C for 60 min, then 45°C for 60 min, hold at 4°C | |
| 30 | Purify DNA with AMPure XP beads (2× ratio), elute in 37.5 μl nuclease-free water | |
| 31 | Use 37 μl eluted DNA for pull-down sample and save 0.5 μl as input sample (add 0.5 μl nuclease-free water, store at -80°C) | |
| 32 | Prepare biotin labeling reaction: 37 μl DNA sample, 5 μl NEBuffer 3.0 (10×), 1 μl 50 mM NAD+, 2 μl 5 μM dATP/dGTP/biotin-dUTP and 20 μM d5fCTP, 1 μl UDG, 1.5 μl Endo IV, 0.8 μl Bst full-length polymerase, 1.7 μl Taq DNA ligase | |
| 33 | Pipette to mix and incubate in thermocycler at 37°C for 40 min, then hold at 4°C | 40 min |
| 34 | Purify DNA with AMPure XP beads (2× ratio), elute in 36 μl nuclease-free water | |
| 35 | Pre-warm malononitrile at 37°C until melted | |
| 36 | Freshly prepare malononitrile solution: 32 μl nuclease-free water, 4 μl 1 M Tris-HCl (pH 7.0), 4 μl melted malononitrile | |
| 37 | Set up chemical reaction: 36 μl DNA sample, 2 μl 100 mM Tris-HCl (pH 7.0), 2 μl malononitrile solution | |
| 38 | Mix thoroughly and incubate at 37°C for 20 h in thermomixer with shaking at 850 rpm (use hot lid) | |
| 39 | Purify DNA with AMPure XP beads (2× ratio), elute in 40 μl nuclease-free water | |
| 40 | Wash Dynabeads MyOne streptavidin C1 (10 μl per sample) three times with 1 ml 1× B&W buffer by gentle rotation (3-5 min each wash) | |
| 41 | Resuspend washed beads in 2× B&W buffer (40 μl per sample) | |
| 42 | Mix 40 μl pre-washed streptavidin C1 beads with 40 μl DNA from Step 39 in 1.5 ml microcentrifuge tube | |
| 43 | Incubate at RT for 1 h with gentle rotation | |
| 44 | Place mixture on magnetic stand, pipette out and discard supernatant | |
| 45 | Wash beads three times with 1 ml 1× B&W buffer | |
| 46 | Resuspend beads in 200 μl 10 mM Tris-HCl (pH 8.0) | |
| 47 | Thaw 30 μM Y-adapter storage solution on ice and dilute to 1.5 μM working solution using 10 mM Tris-HCl (pH 8.0) | |
| 48 | Prepare ligation mix: 14 μl nuclease-free water, 5 μl NEB quick ligation buffer, 2.5 μl 1.5 μM Y-adapter working solution | |
| 49 | For pull-down samples: discard Tris-HCl buffer, add 21.5 μl ligation mix to bead pellet and mix well | |
| 50 | For input samples: add 21.5 μl ligation mix to input DNA and mix well | |
| 51 | Add 2.5 μl Quick T4 DNA ligase and mix well | |
| 52 | For pull-down samples: incubate with gentle rotation at RT for 1 h, add 50 μl 1× B&W buffer, incubate another 1 h | |
| 53 | Wash beads three times with 1 ml 1× B&W buffer, resuspend in 200 μl 1× SSC buffer | |
| 54 | For input samples: incubate ligation reaction at 20°C for 40 min, purify with AMPure XP beads (1× ratio), elute in 22 μl nuclease-free water | 40 min |
| 55 | Discard SSC buffer and resuspend pull-down samples in 20 μl freshly prepared 0.15 M NaOH | |
| 56 | Incubate with gentle rotation at RT for 10 min | |
| 57 | Discard NaOH and resuspend beads in 50 μl 1× SSC buffer | |
| 58 | Wash with 50 μl 10 mM Tris-HCl (pH 8.0) | |
| 59 | Resuspend beads in 22 μl nuclease-free water and heat at 95°C for 3 min (optional: shake at 1200 rpm) | 3 min |
| 60 | Place on magnetic stand and transfer supernatant to clean PCR tube | |
| 61 | Set up first PCR reaction: 22 μl DNA sample, 1 μl NEBNext universal PCR primer (20 μM), 1 μl NEBNext index n primer (20 μM), 25 μl 2× MightyAmp Buffer Ver.3, 1 μl MightyAmp DNA polymerase Ver.3 | |
| 62 | Perform PCR: 98°C for 2 min; 2 cycles of 98°C for 10 s, 68°C for 75 s; hold at 4°C | |
| 63 | Add 1 μl carrier RNA to pull-down samples | |
| 64 | Purify DNA with Vistech DNA Clean & Concentrator 5 kit, elute with 22.5 μl nuclease-free water | |
| 65 | Set up second PCR reaction: 22.5 μl DNA sample, 1.25 μl NEBNext universal PCR primer (20 μM), 1.25 μl NEBNext index n primer (20 μM), 25 μl Q5 Hot Start High-Fidelity 2× mix | |
| 66 | Perform PCR: 98°C for 30 s; n cycles of 98°C for 10 s, 65°C for 90 s; extension at 65°C for 5 min; hold at 4°C (6-7 cycles for input, 8-9 cycles for pull-down) | |
| 67 | Purify DNA with AMPure XP beads (0.9× ratio), elute in 20 μl nuclease-free water | |
| 68 | Measure concentration of final libraries using Qubit fluorometer | |
| 69 | Perform qPCR on spike-in model sequences to check biotin-dUTP incorporation: 10 μl TB Green Premix Ex Taq II, 0.4 μl forward qPCR primer (10 μM), 0.4 μl reverse qPCR primer (10 μM), 1 μl diluted sample, 8.2 μl nuclease-free water | |
| 70 | Perform qPCR: 95°C for 30 s; 40 cycles of 95°C for 5 s, 60°C for 30 s; melting curve | |
| 71 | Calculate enrichment level of modification-containing spike-ins vs control GC sequence using 2^-ΔΔCt method | |
| 72 | Perform PCR on spike-in sequences to check d5fCTP incorporation: 10 μl ZymoTaq Premix, 0.8 μl forward PCR primer (10 μM), 0.8 μl reverse PCR primer (10 μM), 2 μl sample, 6.4 μl nuclease-free water | |
| 73 | Perform PCR: 95°C for 10 min; 32-35 cycles of 95°C for 30 s, 57°C for 30 s, 72°C for 30 s; extension 72°C for 5 min; hold at 4°C | |
| 74 | Subject PCR products to Sanger sequencing to verify tandem C-to-T mutation signals | |
| 75 | Visualize fragment distribution by running 2 ng libraries on Agilent Bioanalyzer or Fragment Analyzer | |
| 76 | Perform paired-end sequencing on Illumina or other massively parallel DNA sequencer (~10-15 million unique paired reads for high-efficiency off-targets, ~60 million for comprehensive detection) | Variable |
| Name | Vendor | Catalog |
|---|---|---|
| CWBIO universal genomic DNA kit | CWBIO | Cat#CW2298 |
| UltraPure DNase/RNase-free distilled water | nuclease-free water | |
| Ethanol | ||
| Vistech DNA Clean & Concentrator-5 kit | Vistech | Cat#DC2005 |
| NEBNext end repair module | New England BioLabs | Cat#E6050S |
| 50 mM NAD+ | ||
| E. coli DNA ligase | New England BioLabs | Cat#M0205S |
| AMPure XP | Beckman Coulter | Cat#A63800 |
| EtONH2 (O-ethylhydroxylamine) | ||
| MES | ||
| NEBNext dA-tailing module | New England BioLabs | Cat#E6053S |
| Klenow fragment (3'-5' exo-) | ||
| 2.5 mM dNTP | Invitrogen | Cat#R72501 |
| NEBNext Endo IV | New England BioLabs | Cat#M0304S |
| NEBuffer 3.0 | New England BioLabs | Cat#B7003S |
| Bst polymerase, full length | New England BioLabs | Cat#M0328S |
| Taq DNA ligase | New England BioLabs | Cat#M0208S |
| dATP | ||
| dGTP | ||
| Biotin-16-aminoallyl-2'-dUTP | TriLink | Cat#N-5001-050 |
| d5fCTP (5-formyl-deoxycytidine triphosphate) | ||
| UDG (Uracil DNA glycosylase) | ||
| 1 M Tris-HCl (pH 7.0) | ||
| Malononitrile | ||
| EDTA | ||
| NaCl | ||
| Tween-20 | CAS no. 9005-64-5; Sigma-Aldrich | Cat#P9416-50ML |
| Dynabeads MyOne streptavidin C1 | ||
| NEBNext quick ligation module | New England BioLabs | Cat#E6056S |
| Quick T4 DNA ligase | New England BioLabs | Cat#B6057AVIAL |
| NaOH | ||
| 20× SSC | Invitrogen | Cat#AM9770 |
| NEBNext index n primer | ||
| MightyAmp DNA polymerase Ver. 3 | TaKaRa | Cat#R076A |
| Carrier RNA | Qiagen | Cat#1068337 |
| NEBNext Q5 Hot Start HiFi PCR master mix | New England BioLabs | Cat#M0543L |
| TBGreen Premix Ex Taq II (Tli RNaseH Plus) | ||
| ZymoTaq Premix | Zymo Research | Cat#E2004 |
| Quibit dsDNA HS assay kits | Invitrogen | Cat#Q32851 |
| D1000 reagents | Agilent | Cat#5067-5583 |
| D1000 ScreenTape | Agilent | Cat#5067-5582 |
| PCR and qPCR primers | Supplementary Table 1 | |
| Spike-in model sequences | ||
| Covaris microTUBE AFA Fiber Pre-Slit Snap-Cap 6×16 mm | ||
| Covaris focused-ultrasonicator ME220 | Covaris | Cat#500506 |
| T100 thermocycler | Bio-Rad | Cat#1861096 |
| Eppendorf ThermoMixer C | Eppendorf | Cat#5382000023 |
| LightCycler 96 instrument | Roche | Cat#05815916001 |
| LightCycler 480 multiwell plate 96, white | Roche | Cat#04729692001 |
| 4150 TapeStation system | Fragment Analyzer | |
| Qubit assay tubes | Invitrogen | Cat#Q32856 |
| Qubit 4 fluorometer | ||
| MagneSphere Technology Magnetic Separation Stands, 1.5 ml | Promega | Cat#Z5342 |
| DiaMag 0.2-ml magnetic rack | Diagenode | Cat#B04000001 |
| NanoDrop |
Zhixin Lei et al. "Detect-seq, a chemical labeling and biotin pull-down approach for the unbiased and genome-wide off-target evaluation of programmable cytosine base editors" Nature Protocols (2023) https://doi.org/10.1038/s41596-023-00837-4 DOI ↗
Direct package: protocol.json