GUIDE-seq library preparation

invivoOfftargetNGSGene editing

21 steps · 13 materials & instruments

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Steps

#	Action	Timing
1	Load 400 ng quantified gDNA into Covaris DNA microTUBE tube with TE buffer to 130 μl total volume
2	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
3	Purify sheared DNA using 1× volume (130 μl) of AMPure XP beads, elute in 15 μl TE buffer
4	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
5	Perform thermocycler program: 12 °C 15 min, 37 °C 15 min, 72 °C 15 min
6	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
7	Perform ligation: 16 °C 30 min, 22 °C 30 min, 4 °C hold
8	Purify ligated product using 0.9× volumes (23 μl) of AMPure XP beads, elute in 22 μl TE buffer
9	Split adapter ligated product for two PCR1 reactions, 10 μl each; label one Plus, one Minus
10	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
11	Assemble Minus PCR1: same as Plus but with oGS202 primer instead of oGS201
12	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	8 h total for library prep
13	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
14	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
15	Assemble Minus PCR2: same as Plus but with oGS205 primer instead of oGS204
16	Run PCR2 thermocycler program: same as PCR1
17	Purify PCR2 product using 0.7× volumes (21 μl) of AMPure XP beads, elute in 30 μl TE buffer
18	Prepare tenfold serial dilutions of libraries from 10^-1 to 10^-6 with TE buffer
19	Assemble SYBR qPCR reactions: combine water 4 μl, diluted template or standard 4 μl, 2× KAPA qPCR MM 12 μl to 20 μl total
20	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
21	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

Materials & instruments

Name	Vendor	Catalog
1× Tris-EDTA (TE) buffer	Fisher Scientific	Cat#AM9848
AMPure XP PCR purification kit	Beckman Coulter	Cat#A63881
End-repair mix	Fisher Scientific	Cat#50305934
T4 DNA ligase	Fisher Scientific	Cat#50305904
25 mM dNTP solution mix	Fisher Scientific	Cat#50305950
Taq polymerase (non-hot start)
Platinum Taq polymerase	Fisher Scientific	Cat#10966-026
Tetramethylammonium chloride (TMAC) 5 M	Sigma	Cat#T3411-500ML
KAPA Library Quantification Kit Universal qPCR Mix	Kapa	Cat#KK4824
Custom Y-adapter oligonucleotides oGS100-oGS116
Acoustic focused ultrasonicator (Covaris E220)
Veriti 96-well thermocycler
CFX96 real-time PCR thermocycler

Direct package: protocol.json