{
 "format": "listen-step-protocol@1",
 "name": "PEM-seq library preparation",
 "tags": [
  "Off-target",
  "Invivo",
  "NGS",
  "Chromesome translocation",
  "Gene editing"
 ],
 "steps": [
  {
   "order": 1,
   "action": "Turn on Covaris M220 focused-ultrasonicator and pre-cool to 4°C",
   "timing": "2 h",
   "materials": [
    "M220 Focused-ultrasonicator"
   ]
  },
  {
   "order": 2,
   "action": "Transfer 20–50 μg DNA to MicroTUBE-130 and adjust volume to 130 μL with nuclease-free water",
   "timing": "2 h",
   "materials": [
    "Nuclease-free water",
    "MicroTUBE-130"
   ]
  },
  {
   "order": 3,
   "action": "Set Covaris M220 parameters: Temperature 4°C, Peak Power 50 W, Duty Factor 20%, Treatment Time 50–65 sec, Cycles 200",
   "timing": "2 h",
   "materials": [
    "M220 Focused-ultrasonicator"
   ]
  },
  {
   "order": 4,
   "action": "Fragment DNA to target peak at 300–700 bp by sonication",
   "timing": "2 h",
   "materials": [
    "M220 Focused-ultrasonicator"
   ]
  },
  {
   "order": 5,
   "action": "Run 1 μL sonicated DNA on 1% (wt/vol) agarose gel in 1× TAE buffer to verify size distribution",
   "timing": "2 h",
   "materials": [
    "Agarose",
    "TAE buffer",
    "Trans DNA Marker II"
   ]
  },
  {
   "order": 6,
   "action": "Prepare primer anneal mixture on ice: 16 μL 10× Bst buffer, 4 μL Bio-primer-MYC1 (+) (1 μM), 32 μL 5 M Betaine, 1–40 μg sonicated DNA, nuclease-free water to 160 μL",
   "timing": "1.5 h",
   "materials": [
    "Bst 3.0 DNA Polymerase buffer",
    "Bio-primer-MYC1",
    "Betaine"
   ]
  },
  {
   "order": 7,
   "action": "Aliquot primer anneal mixtures into 4 PCR tubes",
   "timing": "1.5 h",
   "materials": [
    "0.2 mL PCR strip tubes"
   ]
  },
  {
   "order": 8,
   "action": "Perform primer anneal: 95°C 3 min, then 5 cycles (95°C 2 min, 58°C 3 min), final 58°C 3 min, hold 10°C",
   "timing": "1.5 h",
   "materials": [
    "PCR machine"
   ]
  },
  {
   "order": 9,
   "action": "Prepare primer extension mixture: 4 μL 10× Bst buffer, 4 μL dNTPs (2.5 mM each), 2.5 μL Bst 3.0 DNA polymerase (8 U/μL), 29.5 μL ddH2O",
   "timing": "1.5 h",
   "materials": [
    "Bst 3.0 DNA Polymerase",
    "dNTPs"
   ]
  },
  {
   "order": 10,
   "action": "Add 10 μL primer extension mixture to each PCR tube and mix by vortex",
   "timing": "1.5 h",
   "materials": []
  },
  {
   "order": 11,
   "action": "Perform primer extension: 65°C 15 min, 80°C 5 min (inactivation), hold 25°C",
   "timing": "1.5 h",
   "materials": [
    "PCR machine"
   ]
  },
  {
   "order": 12,
   "action": "Place AMPure XP beads at room temperature for at least 30 min before use",
   "timing": "40 min",
   "materials": [
    "AMPure XP"
   ]
  },
  {
   "order": 13,
   "action": "Add 50 μL pre-warmed AMPure XP beads to each PCR tube, pipette up/down 15–20 times, incubate at room temperature for 5 min",
   "timing": "40 min",
   "materials": [
    "AMPure XP"
   ]
  },
  {
   "order": 14,
   "action": "Place PCR tubes on magnetic stand (DynaMa-PCR Magnet) at room temperature for 5 min",
   "timing": "40 min",
   "materials": [
    "DynaMa-PCR Magnet"
   ]
  },
  {
   "order": 15,
   "action": "Remove and discard supernatant completely",
   "timing": "40 min",
   "materials": []
  },
  {
   "order": 16,
   "action": "Add 200 μL 70% ethanol, remove supernatant completely (repeat twice)",
   "timing": "40 min",
   "materials": [
    "Ethanol"
   ]
  },
  {
   "order": 17,
   "action": "Add 50 μL 10 mM Tris-HCl, pH 7.5, pipette up/down 15–20 times, incubate 2 min",
   "timing": "40 min",
   "materials": [
    "Tris-HCl"
   ]
  },
  {
   "order": 18,
   "action": "Place on magnetic stand 2 min, transfer 50 μL supernatant to new 1.5 mL tube, pool same samples together",
   "timing": "40 min",
   "materials": [
    "Tris-HCl",
    "1.5 mL Microtubes"
   ]
  },
  {
   "order": 19,
   "action": "Check concentration with spectrophotometer to confirm recovery",
   "timing": "40 min",
   "materials": [
    "Spectrophotometer"
   ]
  },
  {
   "order": 20,
   "action": "Incubate purified DNA at 95°C for 5 min and immediately chill on ice for 3 min",
   "timing": "5 h",
   "materials": []
  },
  {
   "order": 21,
   "action": "Prepare streptavidin binding mixture: 200 μL denatured DNA, 50 μL 5 M NaCl, 2.5 μL 0.5 M EDTA, 0.5 μL 10% Triton X-100 (total 253 μL)",
   "timing": "5 h",
   "materials": [
    "NaCl",
    "EDTA",
    "Triton X-100"
   ]
  },
  {
   "order": 22,
   "action": "Transfer 20 μL streptavidin C1 beads to new 1.5 mL tube",
   "timing": "5 h",
   "materials": [
    "Dynabeads MyOne streptavidin C1 beads"
   ]
  },
  {
   "order": 23,
   "action": "Add 400 μL 1× B&W buffer, mix by pipetting, place on DynaMag-2 1 min, remove supernatant (repeat twice)",
   "timing": "5 h",
   "materials": [
    "DynaMag-2",
    "B&W buffer"
   ]
  },
  {
   "order": 24,
   "action": "Suspend C1 beads with 20 μL 1× B&W buffer",
   "timing": "5 h",
   "materials": [
    "Dynabeads MyOne streptavidin C1 beads"
   ]
  },
  {
   "order": 25,
   "action": "Add 20 μL washed streptavidin C1 beads to binding mixture and incubate on rotator at room temperature for 4 h",
   "timing": "4 h",
   "materials": [
    "Dynabeads MyOne streptavidin C1 beads",
    "VWR tube rotator"
   ]
  },
  {
   "order": 26,
   "action": "Spin mixture at 200 g for 5 s (must be <3000 rpm)",
   "timing": "5 h",
   "materials": [
    "Centrifuge"
   ]
  },
  {
   "order": 27,
   "action": "Place on DynaMag-2 holder 1 min, remove supernatant completely",
   "timing": "5 h",
   "materials": [
    "DynaMag-2"
   ]
  },
  {
   "order": 28,
   "action": "Wash C1 beads with 400 μL 1× B&W buffer, capture on magnet 1 min, discard supernatant (repeat twice)",
   "timing": "5 h",
   "materials": [
    "DynaMag-2",
    "B&W buffer"
   ]
  },
  {
   "order": 29,
   "action": "Resuspend beads with 400 μL 10 mM NaOH, immediately place on magnet 1 min, discard supernatant (incubation <2 min)",
   "timing": "5 h",
   "materials": [
    "NaOH",
    "DynaMag-2"
   ]
  },
  {
   "order": 30,
   "action": "Wash beads with 400 μL 10 mM Tris-HCl pH 7.5, capture on magnet 1 min, discard supernatant (repeat twice)",
   "timing": "5 h",
   "materials": [
    "Tris-HCl",
    "DynaMag-2"
   ]
  },
  {
   "order": 31,
   "action": "Resuspend C1 beads with 42.4 μL 10 mM Tris-HCl, pH 7.5",
   "timing": "5 h",
   "materials": [
    "Tris-HCl"
   ]
  },
  {
   "order": 32,
   "action": "Prepare bridge adapter mixture: 20 μL Bridge adapter-upper (400 μM) + 20 μL Bridge adapter-lower (400 μM)",
   "timing": "5 h",
   "materials": [
    "Bridge adapter-upper",
    "Bridge adapter-lower"
   ]
  },
  {
   "order": 33,
   "action": "Anneal bridge adapter: 95°C 3 min, then ramp 85°C to 30°C at 0.1°C/s with 1 min holds, hold 10°C",
   "timing": "5 h",
   "materials": [
    "PCR machine"
   ]
  },
  {
   "order": 34,
   "action": "Add 120 μL nuclease-free water to bring concentration to 50 μM, prepare 80 μL aliquots, store at -20°C",
   "timing": "5 h",
   "materials": [
    "Nuclease-free water"
   ]
  },
  {
   "order": 35,
   "action": "Set up 80 μL ligation: 42.4 μL ssDNA on C1 beads, 8 μL 10× T4 ligase buffer, 1.6 μL Bridge adapter (50 μM), 4 μL T4 DNA ligase (400 U/μL), 24 μL 50% PEG8000",
   "timing": "5 h",
   "materials": [
    "T4 DNA ligase",
    "T4 DNA ligase buffer",
    "PEG8000",
    "Bridge adapter"
   ]
  },
  {
   "order": 36,
   "action": "Mix well (except PEG8000), add PEG8000 with cut-off P200 tip, mix by pipetting 10–15 times",
   "timing": "5 h",
   "materials": [
    "PEG8000"
   ]
  },
  {
   "order": 37,
   "action": "Incubate ligation reaction at room temperature for 4 h or overnight (12–16 h) on rotator at 8 rpm",
   "timing": "4 h or overnight",
   "materials": [
    "VWR tube rotator"
   ]
  },
  {
   "order": 38,
   "action": "Add 320 μL 1× B&W buffer, capture beads on magnet 1 min, remove supernatant",
   "timing": "5 h",
   "materials": [
    "B&W buffer",
    "DynaMag-2"
   ]
  },
  {
   "order": 39,
   "action": "Wash beads with 400 μL 1× B&W buffer (repeat twice), then 10 mM Tris-HCl pH 7.5 (repeat twice)",
   "timing": "5 h",
   "materials": [
    "B&W buffer",
    "Tris-HCl",
    "DynaMag-2"
   ]
  },
  {
   "order": 40,
   "action": "Resuspend on-beads ligated DNA with 73 μL 10 mM Tris-HCl, pH 7.5",
   "timing": "5 h",
   "materials": [
    "Tris-HCl"
   ]
  },
  {
   "order": 41,
   "action": "Set up 100 μL nested PCR: 73 μL on-beads ligation products, 10 μL 10× EasyTaq buffer, 8 μL dNTPs (2.5 mM), 4 μL I5-Nested-1-MYC1 (+) (10 μM), 4 μL I7-index primer (10 μM), 1 μL EasyTaq DNA polymerase (5 U/μL)",
   "timing": "1.5 h",
   "materials": [
    "EasyTaq DNA Polymerase",
    "dNTPs",
    "I5-Nested-1-MYC1",
    "I7-Index primer"
   ]
  },
  {
   "order": 42,
   "action": "Aliquot nested PCR mixture into 2 PCR tubes and perform: 95°C 5 min, then 15 cycles (95°C 1 min, 58°C 45 s, 72°C 1 min), 72°C 5 min, hold 10°C",
   "timing": "1.5 h",
   "materials": [
    "PCR machine"
   ]
  },
  {
   "order": 43,
   "action": "Place AMPure XP beads at room temperature for at least 30 min",
   "timing": "40 min",
   "materials": [
    "AMPure XP"
   ]
  },
  {
   "order": 44,
   "action": "Add 40 μL pre-warmed AMPure XP beads to each PCR tube, pipette 15–20 times, incubate 5 min",
   "timing": "40 min",
   "materials": [
    "AMPure XP"
   ]
  },
  {
   "order": 45,
   "action": "Place on magnetic stand 5 min, remove supernatant, wash with 200 μL 70% ethanol twice",
   "timing": "40 min",
   "materials": [
    "DynaMa-PCR Magnet",
    "Ethanol"
   ]
  },
  {
   "order": 46,
   "action": "Add 35 μL 10 mM Tris-HCl pH 7.5, incubate 2 min, transfer 33 μL supernatant to 1.5 mL tube, pool samples",
   "timing": "40 min",
   "materials": [
    "Tris-HCl"
   ]
  },
  {
   "order": 47,
   "action": "Check concentration with spectrophotometer (should be 1–3 ng/μL)",
   "timing": "40 min",
   "materials": [
    "Spectrophotometer"
   ]
  },
  {
   "order": 48,
   "action": "Set up 100 μL tagged PCR: 63 μL PCR products, 20 μL 5× FastPfu buffer, 8 μL dNTPs (2.5 mM), 4 μL P5-I5 (10 μM), 4 μL P7-tag (10 μM), 1 μL FastPfu DNA polymerase (2.5 U/μL)",
   "timing": "1 h",
   "materials": [
    "FastPfu DNA Polymerase",
    "dNTPs",
    "P5-I5",
    "P7-Tag"
   ]
  },
  {
   "order": 49,
   "action": "Aliquot into 2 PCR tubes, perform: 95°C 2 min, then 10–15 cycles (95°C 30 s, 58°C 45 s, 72°C 1 min), 72°C 5 min, hold 10°C",
   "timing": "1 h",
   "materials": [
    "PCR machine"
   ]
  },
  {
   "order": 50,
   "action": "Pool same samples, add 20 μL 6× DNA loading buffer, run on 1.5% agarose gel in 1× TAE buffer",
   "timing": "3 h",
   "materials": [
    "Agarose",
    "TAE buffer",
    "6× DNA loading buffer"
   ]
  },
  {
   "order": 51,
   "action": "Excise gel with DNA fragments 300–700 bp, cut into small pieces, transfer to 15 mL tube",
   "timing": "3 h",
   "materials": []
  },
  {
   "order": 52,
   "action": "Add 2 mL binding buffer, incubate at 56°C until gel dissolved",
   "timing": "3 h",
   "materials": [
    "GeneJET Gel Extraction Kit"
   ]
  },
  {
   "order": 53,
   "action": "Transfer through column at 10,000 g for 1 min, discard flow-through",
   "timing": "3 h",
   "materials": [
    "GeneJET Gel Extraction Kit"
   ]
  },
  {
   "order": 54,
   "action": "Add 800 μL wash buffer, spin at 10,000 g for 1 min, discard flow-through",
   "timing": "3 h",
   "materials": [
    "GeneJET Gel Extraction Kit"
   ]
  },
  {
   "order": 55,
   "action": "Spin at 10,000 g for 2 min, transfer to new 1.5 mL microtube",
   "timing": "3 h",
   "materials": [
    "1.5 mL Microtubes"
   ]
  },
  {
   "order": 56,
   "action": "Add 50 μL nuclease-free water, incubate 2 min, spin at 10,000 g for 2 min, collect eluted fraction",
   "timing": "3 h",
   "materials": [
    "Nuclease-free water"
   ]
  }
 ],
 "materials": [
  {
   "name": "Nuclease-free water",
   "vendor": "Milli-Q,0.22mmfiltered",
   "catalog": "N/A",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "1 M Tris-HCl, pH 7.5",
   "vendor": "Invitrogen",
   "catalog": "Cat#15567027",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "5 M NaCl",
   "vendor": "Sigma-Aldrich",
   "catalog": "Cat#S6546",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "0.5 M EDTA, pH 8.0",
   "vendor": "Invitrogen",
   "catalog": "Cat#15575020",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Ethanol",
   "vendor": "Sigma-Aldrich",
   "catalog": "Cat#M3148",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Bst 3.0 DNA Polymerase",
   "vendor": "NewEnglandBioLabs",
   "catalog": "Cat#M0374L",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "dNTPs, 2.5 mM each",
   "vendor": "TransGenBiotech",
   "catalog": "Cat#AD101-01",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Betaine solution, 5M",
   "vendor": "Sigma-Aldrich",
   "catalog": "Cat#B0300",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Triton X-100",
   "vendor": "Sigma-Aldrich",
   "catalog": "Cat#T8787",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "10× T4 DNA ligase buffer",
   "vendor": "ThermoScientific",
   "catalog": "Cat#EL0011",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "T4 DNA ligase",
   "vendor": "ThermoScientific",
   "catalog": "Cat#EL0011",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "PEG8000",
   "vendor": "Sigma-Aldrich",
   "catalog": "Cat#89510",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "EasyTaq DNA Polymerase",
   "vendor": "TransGenBiotech",
   "catalog": "Cat#AP111-01",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "AMPure XP",
   "vendor": "BeckmanCoulter",
   "catalog": "Cat#A63880",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "FastPfu DNA Polymerase",
   "vendor": "TransGenBiotech",
   "catalog": "Cat#AP221-01",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Agarose",
   "vendor": "ThermoScientific",
   "catalog": "Cat#75510019",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Trans DNA Marker II",
   "vendor": "TransGenBiotech",
   "catalog": "Cat#BM411-01",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "6× DNA loading buffer",
   "vendor": "BeyotimeBiotech",
   "catalog": "Cat#D0072",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "50× TAE buffer",
   "vendor": "ThermoScientific",
   "catalog": "Cat#B49",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "GeneJET Gel Extraction Kit",
   "vendor": "ThermoScientific",
   "catalog": "Cat#K0692",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Dynabeads MyOne streptavidin C1 beads",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Bio-primer-MYC1 (+)",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Bridge adapter-upper",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Bridge adapter-lower",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "I5-Nested-1-MYC1 (+)",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "P5-I5",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "P7-Tag",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Centrifuge",
   "vendor": "Eppendorf",
   "catalog": "Cat#5406000291",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Spectrophotometer",
   "vendor": "DeNovix",
   "catalog": "Cat#DS-11FX+",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "M220 Focused-ultrasonicator",
   "vendor": "Covaris",
   "catalog": "Cat#500295",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "MicroTUBE-130 AFA Fiber",
   "vendor": "Covaris",
   "catalog": "Cat#520045",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "PCR machine",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "DynaMag-2",
   "vendor": "TransGenBiotech",
   "catalog": "Cat#BM411-01",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "DynaMa-PCR Magnet",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "VWR tube rotator UK plug",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "1.5 mL Microtubes",
   "vendor": "Covaris",
   "catalog": "Cat#520045",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "0.2 mL PCR strip tubes",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  }
 ]
}