{
 "format": "listen-step-protocol@1",
 "name": "GUIDE-seq library preparation",
 "tags": [
  "invivo",
  "Offtarget",
  "NGS",
  "Gene editing"
 ],
 "steps": [
  {
   "order": 1,
   "action": "Load 400 ng quantified gDNA into Covaris DNA microTUBE tube with TE buffer to 130 μl total volume",
   "timing": "",
   "materials": [
    "gDNA",
    "TE buffer",
    "Covaris microTUBE"
   ]
  },
  {
   "order": 2,
   "action": "Shear gDNA to average size of 500 bp using Covaris E220 settings: peak incident power 105 W, duty factor 5%, cycles per burst 200, treatment time 80 s",
   "timing": "",
   "materials": [
    "Covaris E220 ultrasonicator"
   ]
  },
  {
   "order": 3,
   "action": "Purify sheared DNA using 1× volume (130 μl) of AMPure XP beads, elute in 15 μl TE buffer",
   "timing": "",
   "materials": [
    "AMPure XP beads",
    "TE buffer"
   ]
  },
  {
   "order": 4,
   "action": "Assemble end-repair/A-tailing reaction: combine dNTP mix (5 mM each) 1 μl, 10× ligation buffer 2.5 μl, end-repair mix 2 μl, 10× PCR Rxn Buffer 2 μl, Taq polymerase (non-hot start) 0.5 μl, nuclease-free water 0.5 μl, DNA sample 14 μl to 22.5 μl total",
   "timing": "",
   "materials": [
    "End-repair mix",
    "T4 DNA ligase buffer",
    "Taq polymerase",
    "dNTP mix"
   ]
  },
  {
   "order": 5,
   "action": "Perform thermocycler program: 12 °C 15 min, 37 °C 15 min, 72 °C 15 min",
   "timing": "",
   "materials": [
    "Thermocycler"
   ]
  },
  {
   "order": 6,
   "action": "Add 1 μl annealed Y-adapter (oGS100 with one of oGS101-116) and 2 μl T4 DNA ligase to 22.5 μl reaction to 25.5 μl total",
   "timing": "",
   "materials": [
    "Y-adapter",
    "T4 DNA ligase"
   ]
  },
  {
   "order": 7,
   "action": "Perform ligation: 16 °C 30 min, 22 °C 30 min, 4 °C hold",
   "timing": "",
   "materials": [
    "Thermocycler"
   ]
  },
  {
   "order": 8,
   "action": "Purify ligated product using 0.9× volumes (23 μl) of AMPure XP beads, elute in 22 μl TE buffer",
   "timing": "",
   "materials": [
    "AMPure XP beads"
   ]
  },
  {
   "order": 9,
   "action": "Split adapter ligated product for two PCR1 reactions, 10 μl each; label one Plus, one Minus",
   "timing": "",
   "materials": []
  },
  {
   "order": 10,
   "action": "Assemble Plus PCR1: combine water 11.9 μl, Platinum Taq PCR Rxn Buffer 3 μl, dNTP mix 0.6 μl, MgCl2 1.2 μl, oGS201 primer 1 μl, TMAC 1.5 μl, oGS200 primer 0.5 μl, Platinum Taq polymerase 0.3 μl, template DNA 10 μl to 30 μl total",
   "timing": "",
   "materials": [
    "Platinum Taq polymerase",
    "oGS201 primer",
    "oGS200 primer",
    "TMAC"
   ]
  },
  {
   "order": 11,
   "action": "Assemble Minus PCR1: same as Plus but with oGS202 primer instead of oGS201",
   "timing": "",
   "materials": [
    "Platinum Taq polymerase",
    "oGS202 primer",
    "oGS200 primer",
    "TMAC"
   ]
  },
  {
   "order": 12,
   "action": "Run PCR1 thermocycler: 95 °C 5 min; 15 cycles (95 °C 30 s, 70°C Δ-1.0 °C/cycle 2 min, 72 °C 30 s); 10 cycles (95 °C 30 s, 55 °C 1 min, 72 °C 30 s); 72 °C 5 min; 4 °C hold",
   "timing": "8 h total for library prep",
   "materials": [
    "Thermocycler"
   ]
  },
  {
   "order": 13,
   "action": "Purify DNA from each PCR1 reaction using 1.2× volumes (36 μl) of AMPure XP beads, elute in 15 μl TE buffer, keep beads in sample",
   "timing": "",
   "materials": [
    "AMPure XP beads"
   ]
  },
  {
   "order": 14,
   "action": "Assemble Plus PCR2: combine water 5.4 μl, PCR Rxn Buffer 3 μl, dNTP mix 0.6 μl, MgCl2 1.2 μl, oGS204 primer 1 μl, TMAC 1.5 μl, oGS203 primer 0.5 μl, oGS30# primer 1.5 μl, Platinum Taq polymerase 0.3 μl, template DNA 15 μl to 30 μl total",
   "timing": "",
   "materials": [
    "Platinum Taq polymerase",
    "oGS204 primer",
    "oGS203 primer",
    "oGS30# primer",
    "TMAC"
   ]
  },
  {
   "order": 15,
   "action": "Assemble Minus PCR2: same as Plus but with oGS205 primer instead of oGS204",
   "timing": "",
   "materials": [
    "Platinum Taq polymerase",
    "oGS205 primer",
    "oGS203 primer",
    "oGS30# primer",
    "TMAC"
   ]
  },
  {
   "order": 16,
   "action": "Run PCR2 thermocycler program: same as PCR1",
   "timing": "",
   "materials": [
    "Thermocycler"
   ]
  },
  {
   "order": 17,
   "action": "Purify PCR2 product using 0.7× volumes (21 μl) of AMPure XP beads, elute in 30 μl TE buffer",
   "timing": "",
   "materials": [
    "AMPure XP beads",
    "TE buffer"
   ]
  },
  {
   "order": 18,
   "action": "Prepare tenfold serial dilutions of libraries from 10^-1 to 10^-6 with TE buffer",
   "timing": "",
   "materials": [
    "TE buffer"
   ]
  },
  {
   "order": 19,
   "action": "Assemble SYBR qPCR reactions: combine water 4 μl, diluted template or standard 4 μl, 2× KAPA qPCR MM 12 μl to 20 μl total",
   "timing": "",
   "materials": [
    "KAPA qPCR Kit",
    "SYBR Green"
   ]
  },
  {
   "order": 20,
   "action": "Run qPCR program: 95 °C 5 min; 35 cycles (95 °C 30 s, 60 °C 45 s with plate read); melt curve 65-95 °C Δ0.5 °C 5 s",
   "timing": "",
   "materials": [
    "CFX96 real-time PCR thermocycler"
   ]
  },
  {
   "order": 21,
   "action": "Based on calculated concentrations, pool Plus and Minus reactions to achieve equal molecules of each to total 4.5×10^9 molecules in 5 μl for 1.5 nM final concentration",
   "timing": "",
   "materials": []
  }
 ],
 "materials": [
  {
   "name": "1× Tris-EDTA (TE) buffer",
   "vendor": "Fisher Scientific",
   "catalog": "Cat#AM9848",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "AMPure XP PCR purification kit",
   "vendor": "Beckman Coulter",
   "catalog": "Cat#A63881",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "End-repair mix",
   "vendor": "Fisher Scientific",
   "catalog": "Cat#50305934",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "T4 DNA ligase",
   "vendor": "Fisher Scientific",
   "catalog": "Cat#50305904",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "25 mM dNTP solution mix",
   "vendor": "Fisher Scientific",
   "catalog": "Cat#50305950",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Taq polymerase (non-hot start)",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Platinum Taq polymerase",
   "vendor": "Fisher Scientific",
   "catalog": "Cat#10966-026",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Tetramethylammonium chloride (TMAC) 5 M",
   "vendor": "Sigma",
   "catalog": "Cat#T3411-500ML",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "KAPA Library Quantification Kit Universal qPCR Mix",
   "vendor": "Kapa",
   "catalog": "Cat#KK4824",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Custom Y-adapter oligonucleotides oGS100-oGS116",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Acoustic focused ultrasonicator (Covaris E220)",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "Veriti 96-well thermocycler",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  },
  {
   "name": "CFX96 real-time PCR thermocycler",
   "vendor": "",
   "catalog": "",
   "price": "",
   "currency": "",
   "url": ""
  }
 ]
}