Detect-seq library preparation

invitroCBE offtargetNGSgene editing

76 steps · 55 materials & instruments

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Steps

#	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

Materials & instruments

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

Tables

Table 1

DNA sample (~5 μg) from Step 3	Variable
Spike-in model sequences (see Reagent setup; 2.5-10 pg for each)	2
End repair reaction buffer (10×)	10
End repair enzyme mix	5
50 mM NAD+	2
E. coli DNA ligase	2
Nuclease-free water	Up to 100
Total volume	100

Table 2

DNA sample from Step 9	42
dA-tailing reaction buffer	5
Klenow fragment (3′-5′ exo-)	3
Total volume	50

Table 3

DNA sample from Step 12	38
NEBuffer 3.0 (10×)	5
50 mM NAD+	1
2.5 mM dNTPs	1
Endo IV	2
Bst full-length polymerase	1
Taq DNA ligase	2
Total volume	50

Table 4

1	37	60
2	45	60
End	4	Hold

Table 5

DNA sample from Step 15	37
NEBuffer 3.0 (10×)	5
50 mM NAD+	1
5 μM dATP/dGTP/biotin-dUTP/20 μM d5fCTP	2
UDG	1
Endo IV	1.5
Bst full-length polymerase	0.8
Taq DNA ligase	1.7
Total volume	50

Table 6

Nuclease-free water	32
1 M Tris-HCl (pH 7.0)	4
Melted malononitrile	4
Total volume	40

Table 7

DNA sample from Step 18	36
100 mM Tris-HCl (pH 7.0)	2
Malononitrile solution	2
Total volume	40

Table 8

Nuclease-free water	14
NEB quick ligation buffer	5
1.5 μM Y-adapter working solution	2.5
Total volume	21.5

Table 9

DNA sample from Step 31 or 34	22
NEBNext universal PCR primer (20 μM)	1
NEBNext index n primer (20 μM)	1
2× MightyAmp Buffer Ver. 3 ( $Mg^{2+}$ , dNTP plus)	25
MightyAmp DNA polymerase Ver. 3	1
Total volume	50

Table 10

Denaturation	98	2 min
2 cycles	98	10 s
	68	75 s
End	4	Hold

Table 11

DNA sample from Step 37	22.5
NEBNext universal PCR primer (20 μM)	1.25
NEBNext index n primer (20 μM)	1.25
Q5 Hot Start High-Fidelity 2× mix	25
Total volume	50

Table 12

Denaturation	98	30 s
n cycles	98	10 s
	65	90 s
Extension	65	5 min
End	4	Hold

Table 13

TB Green Premix Ex Taq II (Tli RNaseH Plus)	10
Forward qPCR primer (10 μM) (Supplementary Table 1)	0.4
Reverse qPCR primer (10 μM) (Supplementary Table 1)	0.4
Diluted sample	1
Nuclease-free water	8.2
Total volume	20

Table 14

Denaturation	95	30
40 cycles	95	5
	60	30
Melting curve	Instrument's default setting	Instrument's default setting

Table 15

ZymoTaq Premix	10
Forward PCR primer (10 μM) (Supplementary Table 1)	0.8
Reverse PCR primer (10 μM) (Supplementary Table 1)	0.8
Sample	2
Nuclease-free water	6.4
Total volume	20

Table 16

Denaturation	95	10 min
32-35 cycles	95	30 s
	57	30 s
	72	30 s
Extension	72	5 min
End	4	Hold

Table 17

1	The editing efficiency is low	The transfection or delivery method is not efficient enough	Optimize the transfection or delivery reagents and procedure for your editing system
42	The enrichment level is low	The biotin-dUTPs were not efficiently incorporated, or the biotin pull-down step was not well performed	Make sure that no normal dUTPs were used during the biotin labeling step; wash the C1 beads more thoroughly during the biotin pull-down step
43	The tandem C-to-T mutation ratios are low	The d5fCTPs were not efficiently incorporated, or the malononitrile and NaOH treatment was inefficient	Make sure that the concentration of d5fCTPs used is four times higher than that of other dNTPs and that no normal dCTPs were used during the biotin labeling step; ensure that the malononitrile is well dissolved and protected from light during the treatment; use freshly prepared NaOH and incubate with gentle rotation
44	The presence of adapter dimers or the peak of spike-ins is significantly higher than the main library	The starting amount of gDNA is too low, or severe sample loss occurred because of NaOH treatment	Redo Detect-seq with more gDNA and more delicately perform the NaOH washing step; or remove these unwanted peaks by AMPure XP bead clean-up as in Step 7 by using a ratio of 0.8-0.9× beads to sample (this will, to some extent, sacrifice the library quality)

Table 18

Data analysis

Cutadapt (version 1.18)Hisat-3n (version 2.2.1-3n-0.0.2)BWA MEM (version 0.7.17)samtools (version 1.15.1)Picard (version 2.27.4)GATK (version 3.8.1)R environment (version 4.2)Bedtools (version 2.28.0)Integrative Genomics Viewer, IGV (version 2.14.0)Python3.9 https://www.python.org/downloads/Python package pysam v0.19.1 https://pysam.readthedocs.io/en/latest/api.htmlPython package Biopython v1.19 https://biopython.org/Detect-seq tools https://github.com/menghaowei/Detect-seqAgilent TapeStation Software https://www.agilent.com.cn/zh-cn/product/automated-electrophoresis/tapestation-systemsLightCycler® Software https://lifescience.roche.com/en_cn/brands/realtime-pcr-overview.html#software

Table 19

Population characteristics	No human participants
Recruitment	No human participants
Ethics oversight	No human participants

Table 20

Data exclusions	No data was excluded.
Replication	We tested experimental conditions with different DdCBE TALE arrays. We also performed biological replicates independently and successfully of each other.
Randomization	Because the sample size is small, randomization was not relevant for this study. Covariates were controlled for by running controls in parallel whenever applicable.
Blinding	Blinding was not performed as experimental conditions were evident.

Table 21

☒	Antibodies
☐	Eukaryotic cell lines
☒	Palaeontology and archaeology
☒	Animals and other organisms
☒	Clinical data
☒	Dual use research of concern

Table 22

☒	☐ ChIP-seq
☒	☐ Flow cytometry
☒	☐ MRI-based neuroimaging

Table 23

Authentication	No cell lines were authenticated
Mycoplasma contamination	Cells tested negative for mycoplasma as detailed in the Methods.
Commonly misidentified lines (See ICLAC register)	No commonly misidentified cell lines were used.

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 DOI ↗

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