{
 "format": "listen-step-protocol@1",
 "name": "Detect-seq library preparation",
 "tags": [
  "invitro",
  "CBE offtarget",
  "NGS",
  "gene editing"
 ],
 "source": {
  "filename": "Detect seq a chemical labeling and Source Nat Protoc SO 2023.pdf",
  "citation": "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",
  "paper": {
   "doi": "10.1038/s41596-023-00837-4",
   "url": "https://doi.org/10.1038/s41596-023-00837-4",
   "year": 2023,
   "title": "Detect-seq, a chemical labeling and biotin pull-down approach for the unbiased and genome-wide off-target evaluation of programmable cytosine base editors",
   "authors": [
    "Zhixin Lei",
    "Haowei Meng",
    "Xichen Rao",
    "Huanan Zhao",
    "Chengqi Yi"
   ],
   "journal": "Nature Protocols"
  }
 },
 "steps": [
  {
   "order": 1,
   "action": "Extract genomic DNA from ~5-10×10^6 edited living cells (HEK293T cells) using CWBIO universal gDNA kit following the manufacturer's instructions",
   "timing": "20-40 min",
   "materials": [
    "CWBIO universal genomic DNA kit"
   ]
  },
  {
   "order": 2,
   "action": "Quantify DNA using NanoDrop spectrophotometer",
   "timing": "",
   "materials": [
    "NanoDrop"
   ]
  },
  {
   "order": 3,
   "action": "Check editing efficiency at on-target sites by Sanger sequencing or targeted amplicon sequencing",
   "timing": "",
   "materials": []
  },
  {
   "order": 4,
   "action": "Load 3.75 μg gDNA into Covaris microTUBE supplemented with 10 mM Tris-HCl (pH 8.0) to total volume of 130 μl",
   "timing": "",
   "materials": [
    "Covaris microTUBE AFA Fiber Pre-Slit Snap-Cap",
    "1 M Tris-HCl (pH 8.0)"
   ]
  },
  {
   "order": 5,
   "action": "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",
   "timing": "70 s",
   "materials": [
    "Covaris focused-ultrasonicator ME220"
   ]
  },
  {
   "order": 6,
   "action": "Fragment another 3.75 μg of quantified gDNA for each sample to obtain ≥5 μg sheared DNA after purification",
   "timing": "",
   "materials": []
  },
  {
   "order": 7,
   "action": "Purify fragmented DNA with Vistech DNA Clean & Concentrator 5 kit using 5 volumes DNA binding buffer per volume of DNA sample",
   "timing": "",
   "materials": [
    "Vistech DNA Clean & Concentrator-5 kit"
   ]
  },
  {
   "order": 8,
   "action": "Elute with 60 μl nuclease-free water at room temperature",
   "timing": "",
   "materials": [
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 9,
   "action": "Quantify DNA using NanoDrop spectrophotometer",
   "timing": "",
   "materials": [
    "NanoDrop"
   ]
  },
  {
   "order": 10,
   "action": "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",
   "timing": "",
   "materials": [
    "NEBNext end repair module",
    "50 mM NAD+",
    "E. coli DNA ligase",
    "UltraPure DNase/RNase-free distilled water",
    "Spike-in model sequences"
   ]
  },
  {
   "order": 11,
   "action": "Pipette on ice to mix thoroughly",
   "timing": "",
   "materials": []
  },
  {
   "order": 12,
   "action": "Incubate in thermocycler at 20°C for 30 min",
   "timing": "30 min",
   "materials": [
    "T100 thermocycler"
   ]
  },
  {
   "order": 13,
   "action": "Purify DNA with AMPure XP beads using ratio of 2× beads to sample (e.g., add 200 μl to 100 μl sample)",
   "timing": "",
   "materials": [
    "AMPure XP"
   ]
  },
  {
   "order": 14,
   "action": "Mix by briefly vortexing and incubate at RT for 10-15 min",
   "timing": "10-15 min",
   "materials": []
  },
  {
   "order": 15,
   "action": "Place tubes on magnetic plate until liquid is clear, then discard supernatant",
   "timing": "",
   "materials": [
    "DiaMag 0.2-ml magnetic rack"
   ]
  },
  {
   "order": 16,
   "action": "Wash beads with 80% (vol/vol) ethanol, 500 μl per wash, incubate 30 s, aspirate and discard",
   "timing": "",
   "materials": [
    "Ethanol"
   ]
  },
  {
   "order": 17,
   "action": "Repeat 80% ethanol wash and air-dry pellet for 5-10 min",
   "timing": "5-10 min",
   "materials": [
    "Ethanol"
   ]
  },
  {
   "order": 18,
   "action": "Resuspend pellet in 40 μl nuclease-free water by pipetting up and down thoroughly",
   "timing": "",
   "materials": [
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 19,
   "action": "Incubate at RT for 5-15 min, then transfer supernatant to new PCR tube",
   "timing": "",
   "materials": [
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 20,
   "action": "Mix 40 μl DNA sample with 40 μl of 2× EtONH2 buffer",
   "timing": "",
   "materials": [
    "EtONH2 (O-ethylhydroxylamine)",
    "MES",
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 21,
   "action": "Incubate at 37°C for 6 h in thermomixer with shaking at 850 rpm",
   "timing": "6 h",
   "materials": [
    "Eppendorf ThermoMixer C"
   ]
  },
  {
   "order": 22,
   "action": "Dilute reaction by adding 120 μl nuclease-free water",
   "timing": "",
   "materials": [
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 23,
   "action": "Purify DNA with Vistech DNA Clean & Concentrator 5 kit",
   "timing": "",
   "materials": [
    "Vistech DNA Clean & Concentrator-5 kit"
   ]
  },
  {
   "order": 24,
   "action": "Elute with 42 μl nuclease-free water at RT",
   "timing": "",
   "materials": [
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 25,
   "action": "Set up dA-tailing reaction: 42 μl DNA sample, 5 μl dA-tailing reaction buffer, 3 μl Klenow fragment (3'-5' exo-)",
   "timing": "",
   "materials": [
    "NEBNext dA-tailing module",
    "Klenow fragment (3'-5' exo-)"
   ]
  },
  {
   "order": 26,
   "action": "Pipette to mix and incubate in thermocycler at 37°C for 30 min",
   "timing": "30 min",
   "materials": [
    "T100 thermocycler"
   ]
  },
  {
   "order": 27,
   "action": "Purify DNA with AMPure XP beads (2× ratio), elute in 38 μl nuclease-free water",
   "timing": "",
   "materials": [
    "AMPure XP",
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 28,
   "action": "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",
   "timing": "",
   "materials": [
    "NEBNext Endo IV",
    "NEBuffer 3.0",
    "50 mM NAD+",
    "2.5 mM dNTP",
    "Bst polymerase, full length",
    "Taq DNA ligase"
   ]
  },
  {
   "order": 29,
   "action": "Pipette to mix and perform reaction in thermocycler: 37°C for 60 min, then 45°C for 60 min, hold at 4°C",
   "timing": "",
   "materials": [
    "T100 thermocycler"
   ]
  },
  {
   "order": 30,
   "action": "Purify DNA with AMPure XP beads (2× ratio), elute in 37.5 μl nuclease-free water",
   "timing": "",
   "materials": [
    "AMPure XP",
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 31,
   "action": "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)",
   "timing": "",
   "materials": [
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 32,
   "action": "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",
   "timing": "",
   "materials": [
    "NEBuffer 3.0",
    "50 mM NAD+",
    "dATP",
    "dGTP",
    "Biotin-16-aminoallyl-2'-dUTP",
    "d5fCTP (5-formyl-deoxycytidine triphosphate)",
    "UDG (Uracil DNA glycosylase)",
    "Endo IV",
    "Bst polymerase, full length",
    "Taq DNA ligase"
   ]
  },
  {
   "order": 33,
   "action": "Pipette to mix and incubate in thermocycler at 37°C for 40 min, then hold at 4°C",
   "timing": "40 min",
   "materials": [
    "T100 thermocycler"
   ]
  },
  {
   "order": 34,
   "action": "Purify DNA with AMPure XP beads (2× ratio), elute in 36 μl nuclease-free water",
   "timing": "",
   "materials": [
    "AMPure XP",
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 35,
   "action": "Pre-warm malononitrile at 37°C until melted",
   "timing": "",
   "materials": [
    "Malononitrile"
   ]
  },
  {
   "order": 36,
   "action": "Freshly prepare malononitrile solution: 32 μl nuclease-free water, 4 μl 1 M Tris-HCl (pH 7.0), 4 μl melted malononitrile",
   "timing": "",
   "materials": [
    "Malononitrile",
    "1 M Tris-HCl (pH 7.0)",
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 37,
   "action": "Set up chemical reaction: 36 μl DNA sample, 2 μl 100 mM Tris-HCl (pH 7.0), 2 μl malononitrile solution",
   "timing": "",
   "materials": [
    "Malononitrile",
    "1 M Tris-HCl (pH 7.0)"
   ]
  },
  {
   "order": 38,
   "action": "Mix thoroughly and incubate at 37°C for 20 h in thermomixer with shaking at 850 rpm (use hot lid)",
   "timing": "",
   "materials": [
    "Eppendorf ThermoMixer C"
   ]
  },
  {
   "order": 39,
   "action": "Purify DNA with AMPure XP beads (2× ratio), elute in 40 μl nuclease-free water",
   "timing": "",
   "materials": [
    "AMPure XP",
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 40,
   "action": "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)",
   "timing": "",
   "materials": [
    "Dynabeads MyOne streptavidin C1",
    "1 M Tris-HCl (pH 7.5)",
    "EDTA",
    "NaCl",
    "Tween-20",
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 41,
   "action": "Resuspend washed beads in 2× B&W buffer (40 μl per sample)",
   "timing": "",
   "materials": [
    "1 M Tris-HCl (pH 7.5)",
    "EDTA",
    "NaCl",
    "Tween-20",
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 42,
   "action": "Mix 40 μl pre-washed streptavidin C1 beads with 40 μl DNA from Step 39 in 1.5 ml microcentrifuge tube",
   "timing": "",
   "materials": [
    "Dynabeads MyOne streptavidin C1"
   ]
  },
  {
   "order": 43,
   "action": "Incubate at RT for 1 h with gentle rotation",
   "timing": "",
   "materials": []
  },
  {
   "order": 44,
   "action": "Place mixture on magnetic stand, pipette out and discard supernatant",
   "timing": "",
   "materials": [
    "MagneSphere Technology Magnetic Separation Stands"
   ]
  },
  {
   "order": 45,
   "action": "Wash beads three times with 1 ml 1× B&W buffer",
   "timing": "",
   "materials": [
    "1 M Tris-HCl (pH 7.5)",
    "EDTA",
    "NaCl",
    "Tween-20"
   ]
  },
  {
   "order": 46,
   "action": "Resuspend beads in 200 μl 10 mM Tris-HCl (pH 8.0)",
   "timing": "",
   "materials": [
    "1 M Tris-HCl (pH 8.0)"
   ]
  },
  {
   "order": 47,
   "action": "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)",
   "timing": "",
   "materials": [
    "1 M Tris-HCl (pH 8.0)"
   ]
  },
  {
   "order": 48,
   "action": "Prepare ligation mix: 14 μl nuclease-free water, 5 μl NEB quick ligation buffer, 2.5 μl 1.5 μM Y-adapter working solution",
   "timing": "",
   "materials": [
    "NEBNext quick ligation module",
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 49,
   "action": "For pull-down samples: discard Tris-HCl buffer, add 21.5 μl ligation mix to bead pellet and mix well",
   "timing": "",
   "materials": [
    "NEBNext quick ligation module"
   ]
  },
  {
   "order": 50,
   "action": "For input samples: add 21.5 μl ligation mix to input DNA and mix well",
   "timing": "",
   "materials": [
    "NEBNext quick ligation module"
   ]
  },
  {
   "order": 51,
   "action": "Add 2.5 μl Quick T4 DNA ligase and mix well",
   "timing": "",
   "materials": [
    "Quick T4 DNA ligase"
   ]
  },
  {
   "order": 52,
   "action": "For pull-down samples: incubate with gentle rotation at RT for 1 h, add 50 μl 1× B&W buffer, incubate another 1 h",
   "timing": "",
   "materials": [
    "NEBNext quick ligation module"
   ]
  },
  {
   "order": 53,
   "action": "Wash beads three times with 1 ml 1× B&W buffer, resuspend in 200 μl 1× SSC buffer",
   "timing": "",
   "materials": [
    "20× SSC"
   ]
  },
  {
   "order": 54,
   "action": "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",
   "timing": "40 min",
   "materials": [
    "AMPure XP",
    "T100 thermocycler",
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 55,
   "action": "Discard SSC buffer and resuspend pull-down samples in 20 μl freshly prepared 0.15 M NaOH",
   "timing": "",
   "materials": [
    "NaOH"
   ]
  },
  {
   "order": 56,
   "action": "Incubate with gentle rotation at RT for 10 min",
   "timing": "",
   "materials": []
  },
  {
   "order": 57,
   "action": "Discard NaOH and resuspend beads in 50 μl 1× SSC buffer",
   "timing": "",
   "materials": [
    "20× SSC"
   ]
  },
  {
   "order": 58,
   "action": "Wash with 50 μl 10 mM Tris-HCl (pH 8.0)",
   "timing": "",
   "materials": [
    "1 M Tris-HCl (pH 8.0)"
   ]
  },
  {
   "order": 59,
   "action": "Resuspend beads in 22 μl nuclease-free water and heat at 95°C for 3 min (optional: shake at 1200 rpm)",
   "timing": "3 min",
   "materials": [
    "UltraPure DNase/RNase-free distilled water",
    "Eppendorf ThermoMixer C"
   ]
  },
  {
   "order": 60,
   "action": "Place on magnetic stand and transfer supernatant to clean PCR tube",
   "timing": "",
   "materials": [
    "DiaMag 0.2-ml magnetic rack"
   ]
  },
  {
   "order": 61,
   "action": "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",
   "timing": "",
   "materials": [
    "MightyAmp DNA polymerase Ver. 3",
    "NEBNext index n primer"
   ]
  },
  {
   "order": 62,
   "action": "Perform PCR: 98°C for 2 min; 2 cycles of 98°C for 10 s, 68°C for 75 s; hold at 4°C",
   "timing": "",
   "materials": [
    "T100 thermocycler"
   ]
  },
  {
   "order": 63,
   "action": "Add 1 μl carrier RNA to pull-down samples",
   "timing": "",
   "materials": [
    "Carrier RNA"
   ]
  },
  {
   "order": 64,
   "action": "Purify DNA with Vistech DNA Clean & Concentrator 5 kit, elute with 22.5 μl nuclease-free water",
   "timing": "",
   "materials": [
    "Vistech DNA Clean & Concentrator-5 kit",
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 65,
   "action": "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",
   "timing": "",
   "materials": [
    "NEBNext Q5 Hot Start HiFi PCR master mix",
    "NEBNext index n primer"
   ]
  },
  {
   "order": 66,
   "action": "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)",
   "timing": "",
   "materials": [
    "T100 thermocycler"
   ]
  },
  {
   "order": 67,
   "action": "Purify DNA with AMPure XP beads (0.9× ratio), elute in 20 μl nuclease-free water",
   "timing": "",
   "materials": [
    "AMPure XP",
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 68,
   "action": "Measure concentration of final libraries using Qubit fluorometer",
   "timing": "",
   "materials": [
    "Quibit dsDNA HS assay kits",
    "Qubit 4 fluorometer",
    "Qubit assay tubes"
   ]
  },
  {
   "order": 69,
   "action": "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",
   "timing": "",
   "materials": [
    "TBGreen Premix Ex Taq II",
    "PCR and qPCR primers",
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 70,
   "action": "Perform qPCR: 95°C for 30 s; 40 cycles of 95°C for 5 s, 60°C for 30 s; melting curve",
   "timing": "",
   "materials": [
    "LightCycler 96 instrument",
    "LightCycler 480 multiwell plate 96, white"
   ]
  },
  {
   "order": 71,
   "action": "Calculate enrichment level of modification-containing spike-ins vs control GC sequence using 2^-ΔΔCt method",
   "timing": "",
   "materials": []
  },
  {
   "order": 72,
   "action": "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",
   "timing": "",
   "materials": [
    "ZymoTaq Premix",
    "PCR and qPCR primers",
    "UltraPure DNase/RNase-free distilled water"
   ]
  },
  {
   "order": 73,
   "action": "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",
   "timing": "",
   "materials": [
    "T100 thermocycler"
   ]
  },
  {
   "order": 74,
   "action": "Subject PCR products to Sanger sequencing to verify tandem C-to-T mutation signals",
   "timing": "",
   "materials": []
  },
  {
   "order": 75,
   "action": "Visualize fragment distribution by running 2 ng libraries on Agilent Bioanalyzer or Fragment Analyzer",
   "timing": "",
   "materials": [
    "D1000 reagents",
    "D1000 ScreenTape",
    "4150 TapeStation system"
   ]
  },
  {
   "order": 76,
   "action": "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)",
   "timing": "Variable",
   "materials": []
  }
 ],
 "materials": [
  {
   "name": "CWBIO universal genomic DNA kit",
   "vendor": "CWBIO",
   "catalog": "Cat#CW2298",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "UltraPure DNase/RNase-free distilled water",
   "vendor": "nuclease-free water",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Ethanol",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Vistech DNA Clean & Concentrator-5 kit",
   "vendor": "Vistech",
   "catalog": "Cat#DC2005",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "NEBNext end repair module",
   "vendor": "New England BioLabs",
   "catalog": "Cat#E6050S",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "50 mM NAD+",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "E. coli DNA ligase",
   "vendor": "New England BioLabs",
   "catalog": "Cat#M0205S",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "AMPure XP",
   "vendor": "Beckman Coulter",
   "catalog": "Cat#A63800",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "EtONH2 (O-ethylhydroxylamine)",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "MES",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "NEBNext dA-tailing module",
   "vendor": "New England BioLabs",
   "catalog": "Cat#E6053S",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Klenow fragment (3'-5' exo-)",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "2.5 mM dNTP",
   "vendor": "Invitrogen",
   "catalog": "Cat#R72501",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "NEBNext Endo IV",
   "vendor": "New England BioLabs",
   "catalog": "Cat#M0304S",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
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