DAY 1
1. Genomic DNA (gDNA) extraction
Note: the following is a procedure to isolate gDNA from small regions within single FFPE tissue sections using the PinPoint Slide DNA Isolation System™. For tissue sections or cells, any isolation protocol yielding high-quality gDNA, including commercially available silica-based kits, is compatible with the subsequent steps of CUTseq.
1. Apply a little amount of PinPoint glue onto a small region of interest of a FFPE tissue section previously stained with hematoxylin-eosin and imaged
Note: this step is recommended to confirm the presence of tumor cells within the tissue region
2. Air-dry the PinPoint glue for at least 30 min at room temperature
3. Cut the solidified glue using a disposable insulin needle, then transfer the pellet into a LoBind tube pre-filled with 100 μl of tail buffer plus 10 μl of proteinase K
4. Incubate on a thermomixer for 18 hours @ 55 °C, shaking at 800 rpm
DAY 2
1. Add 10 μl of fresh proteinase K
2. Incubate for 1 hour @ 55 °C
3. Transfer the sample to a PCR thermocycler, and incubate for 10 min @ 95 °C to inactivate proteinase K
4. Purify gDNA using phenol-chloroform
Note: in our experience, phenol-chloroform extraction yields gDNA of the highest quality. However, we have also successfully used silica columns provided in the PinPoint Slide DNA Isolation System™
5. Add 120 μl of phenol/chloroform pre-warmed at room temperature, and shake vigorously
6. Centrifuge at ~20,000 x g for 5 min at room temperature
7. Collect the upper phase (~100 μl) and transfer it to a new LoBind tube
8. Add an equal volume of chloroform and shake vigorously
9. Centrifuge at ~20,000 x g for 5min at room temperature
10. Collect the upper phase (~60 μl) and transfer it to a new LoBind tube
11. Add 3.7 μl of glycogen per 100 μl of solution, and vortex vigorously to mix
12. Add Na-Acetate 3M, pH 5.5 to a final concentration of 0.3 M, and vortex vigorously to mix
13. Add 2.5 volumes of ice-cold 100% ethanol, then immediately vortex vigorously to mix
14. Incubate for 16–18 hours @ –80 °C
DAY 3
1. Centrifuge at ~20,000 x g for 1 hour @ 4 °C
2. Gently discard the supernatant
3. Wash the DNA pellet with 500 μl of ice-cold 70% ethanol, by vortexing vigorously
4. Centrifuge at ~20,000 x g for 15 min @ 4 °C
5. Discard the supernatant and wash the pellet again with 500 μl of ice-cold 70% ethanol
6. Centrifuge at ~20,000 x g for 15 min @ 4 °C
7. Discard the supernatant
8. Air-dry the DNA pellet
Note: avoid over-drying the DNA pellet, as this may result in low DNA yield
9. Resuspend the pellet in 8 μl of nuclease-free water
Breakpoint: if needed, at this point the samples can be stored @ –20 °C for several months
DAY 3
2. DNA Digestion
Note: here we describe the use of HindIII to digest gDNA, since this enzyme was used for most of the experiments described in this manuscript. However, different restriction enzymes can be used at this point to digest the purified gDNA. The choice of enzyme depends on the application and desired resolution. For example, for applications that require high genome coverage, such as exome sequencing, a 4-cutter is preferable. On the other hand, 6-cutters are better suited for applications for which shallow genome sequencing is sufficient, for instance DNA copy number profiling at low resolution. Additional aspects to consider when choosing which restriction enzyme to use are the distribution of recognition sites along the genome of interest (see Supplementary Figure 1), the sensitivity of different enzymes to methylation in the recognition sequence, as well as the cost of purchasing the enzymes from commercial providers. A list of enzymes that we recommend for CUTseq and are available through NEW ENGLAND BioLabs is provided in Supplementary Table 1.
Standard CUTseq
1. Transfer 8 μl of purified gDNA into a 0.5 ml LoBind tube
Note: when processing multiple samples to be pooled into the same library, PCR tube strips or 96-well plates can be used, depending on the number of samples
2. Add 2 μl of digestion mix:
CutSmart buffer 10X 1 μl
HindIII-HF 1 μl
3. Perform the following steps in a PCR thermocycler with the lid set @ 50 °C:
1. 37 °C 16-18 h
2. 80 °C 20 min
3. 4 °C Hold
High-throughput CUTseq
Note: the following protocol was implemented on the I-DOT One nanodispensing system (Dispendix, Germany). Other systems may also be used; however, volumes might have to be adjusted depending on the technical specifications of each instrument.
# Dispense with I-DOT
1. Dispense 5 μl of Vapor Lock per well in a 96- or 384-well plate
Note: 384-well plates are better suited when dispensing with nanoliter volumes as the conical bottom and size of the wells allows to more easily visualize the dispensed droplets inside the Vapor Lock phase.
2. @ Add 350 nl per well of gDNA per well
3. @ Add 150 nl per well of digestion mix:
CutSmart buffer 10X 50 nl
HindIII-HF 100nl
4. Perform the following steps in a thermo incubator:
1. 37 ° 30 min
2. 80 °C 20 min
3. 4 °C Hold
DAY 4 (DAY 3 for high-throughput CUTseq)
3. Ligation of CUTseq adapters
Standard CUTseq
1. Add 1μl per sample of 0.33 μM HindIII adapter with the desired barcode sequence
2. Place the sample(s) on ice
3. To each sample, add 19 μl of ligation mix:
Nuclease-free water 12 μl
T4 ligase buffer 10X 3 μl
ATP 10 mM 2.4 μl
BSA 50 mg/ml 0.6 μl
T4 ligase highly conc. 1 μl
4. In a PCR thermocycler with the lid set @ 20 °C, perform the following steps:
1. 16 °C 16-18 h
2. 65 °C 10 min
3. 4 °C Hold
High-throughput CUTseq
1. @ Add 300 nl of 33 nM CUTseq adapter per well
2. @ Add 700 nl of ligation mix per well:
Nuclease-free water 50 nl
T4 ligase buffer 5X 300 nl
ATP 10 mM 120 nl
BSA 50 mg/ml 30 nl
T4 rapid ligase 200 nl
3. Incubate at room temperature for 30 min
4. Pool the volume in multiple wells into one 1.5 ml LoBind tube
Note: in order to be pooled together, multiple wells must have different sample barcodes. The number of wells pooled together depends on the total number of samples, and on the desired complexity of the final library. In general, libraries containing many samples will need to be sequenced deeply in order for each sample to receive a sufficient number of reads. In our experience, we have successfully sequenced libraries containing up to 96 different barcodes in a single NextSeq 500 run, obtaining a number of reads per sample sufficient to perform reproducible DNA copy number profiling (see Figure 3).
DAY 5 (DAY 3 for high-throughput CUTseq)
4. DNA cleanup
Note: at this point, the same procedure is followed for single-sample and multiplexed libraries. Below, we describe a standard DNA precipitation cleanup procedure. However, silica-based columns or AMPure XP beads can also be used, according to the manufacturer’s instructions.
1. Measure the sample volume, then add the following reagents:
Glycogen 20 mg/ml 3.7 μl per 100 μl
Na-Acetate 3 M, pH 5.5 Up to 0.3 M final conc.
2. Add 2.5 volumes of ice-cold 100% ethanol
3. Vortex 5 seconds at max. power
4. Incubate for 18 hours @ –80 °C
Note: incubation @ –20 °C is also fine
DAY 6 (DAY 4 for high-throughput CUTseq)
5. Centrifuge at ~20,000 x g for 1 hour @ 4 °C
6. Discard the supernatant
7. Wash the DNA pellet with 500 μl of ice-cold 70% ethanol
8. Centrifuge at ~20,000 x g for 15 min @ 4 °C
9. Discard the supernatant and wash the pellet again with 500 μl of ice-cold 70% ethanol
10. Centrifuge at ~20,000 x g for 15 min @ 4 °C
11. Discard the supernatant
12. Air-dry the pellet
Note: avoid over-drying the DNA pellet, as this may result in low DNA yield
10. Resuspend the pellet in 50 μl of nuclease-free water
Breakpoint: if needed, at this point the samples can be stored at –20 °C for several months
DAY 6 (DAY 4 for high-throughput CUTseq)
5. Sonication
Note: in our experience, DNA fragments of 200–300 bp are needed to obtain high-quality libraries and sequencing results. To sonicate gDNA, we typically use a Covaris ME220 Focused-ultrasonicator with microTUBE-50 AFA Fiber Screw-Cap tubes or microTUBE-15 AFA Beads Screw-Cap tubes, with a target peak of 200 bp. Note that we sonicate gDNA independently of the restriction enzyme used, as well as gDNA extracted from FFPE samples, since we have found that this results in higher-quality libraries.
6. SpeedVac
Transfer sample from covaris tube to new 0.5 ml DNA LoBind tube, proceed to SpeedVac Vaccum Concentrator with a normal heating mode till the moment when the volume of sample is entirely evaporated.
DAY 6 (DAY 4 for high-throughput CUTseq)
7. In vitro transcription
1. Resuspend the sample with 8 μl nuclease-free water
2. Add the following reagents on ice:
rATP+rUTP+rGTP+rCTP* 8 μl
T7 polymerase buffer 10X 2 μl
T7 polymerase 1.5 μl
RNaseOUTTM Recombinant Ribonuclease Inhibitor 40U 0.5 μl
*Prepared from separate rNTP solutions provided with the MEGAscript® T7 Transcription Kit
3. Incubate for 14 hours (for Standard CUTseq) or 2 hours (for high-throughput CUTseq) @ 37 °C in a PCR thermocycler with the lid set @ 70 °C
DAY 7 (DAY 5 for high-throughput CUTseq)
8. RNA cleanup
1. Add 1 μl of DNAse I RNase-free to the IVT product
2. Incubate for 15 min @ 37 °C
3. Bring up the volume to 30 μl by adding 9 μl nuclease-free water, then mix with 54 μl (1.8x) of RNAClean XP beads pre-warmed at room temperature
4. Mix thoroughly and incubate for 10 min at room temperature
5. Place the sample on a magnetic stand
6. Incubate for at least 5 min until the liquid appears clear
7. Remove and discard the supernatant
8. Wash the beads twice with 200 μl of freshly prepared 70% ice-cold ethanol
9. Air-dry the beads at room temperature
Note: do not dry the beads for more than 5–8 min, since this may result in low DNA yield
10. Remove the sample from the magnetic stand
11. Resuspend the beads in 8 μl of nuclease-free water
12. Incubate for 2 min at room temperature
13. Place the sample back on the magnetic stand
14. Incubate for at least 5 min until the liquid appears clear
15. Transfer 7.8 μl of supernatant to a new 0.5 μl DNA LoBind tube
Note: the following steps until section 11. are adapted from the TruSeq Small RNA Library Preparation protocol from Illumina. *Steps on ice.
DAY 7 (DAY 5 for high-throughput CUTseq)
9. RA3 adapter ligation
1. * Add 1 μl of 10 μM RA3 adapter
2. Incubate for 2 min @ 70 °C in a PCR thermocycler, then immediately place sample on ice
3. * Add 3.2 μl of the following mix:
RNA ligase buffer 1.2 μl
RNaseOUTTM Recombinant Ribonuclease Inhibitor 40U 1 μl
T4 RNA ligase truncated 1 μl
4. Incubate for 2 hours @ 25 °C in a PCR thermocycler with the lid set @ 30 °C
DAY 7 (DAY 5 for high-throughput CUTseq)
10. Reverse transcription (1st strand synthesis)
1. * Add 2 μl per sample of RTP primer
2. In a PCR thermocycler, incubate for 2 min @ 70 °C
3. * Quickly transfer the sample to ice
4. * Add 11 μl of the following mix:
1st stand buffer 5 μl
dNTPs @ 12.5 mM 1 μl
100 mM DTT 2 μl
RNaseOUTTM Recombinant Ribonuclease Inhibitor 40 U 1 μl
SuperScript IV reverse transcriptase 2 μl
5. In a PCR thermocycler with the lid set @ 50 °C, perform the following steps:
1. 50 °C 1 hour
2. 80 °C 10 min
3. 4 °C Hold
Breakpoint: if needed, at this point the samples can be stored @ –20 °C for several months
DAY 7 (DAY 5 for high-throughput CUTseq)
11. Library indexing and amplification
1. * Add 4 μl per sample of the desired indexed Illumina primer
2. * Add 71 μl of the following mix:
Nuclease-free water 17 μl
NEBNext® UltraTM II PCR Master Mix 50 μl
RP1 primer 4 μl
3. In a PCR thermocycler perform the following cycles:
1. 98 °C30 sec
2. 98 °C 10 sec
3. 65 °C 75 sec
GOTO step 2, 8-20 times
4. 65 °C 5 min
5. 4 °C Hold
Note: the number of PCR cycles needs to be adjusted depending on the gDNA input in the IVT reaction. The following table shows the recommended number of PCR cycles for various gDNA inputs, based on our experience:
Input gDNA (ng)
# PCR cycles
500-700 ng gDNA input: 6 cycles
300-400 ng gDNA input: 7 cycles
100-200 ng gDNA input: 8 cycles
50-100 ng gDNA input: 9 cycles
30 ng gDNA input: 10 cycles
15 ng gDNA input: 11 cycles
7.5 ng gDNA input: 12 cycles
3.8 ng gDNA input: 13 cycles
1.9 ng gDNA input: 14 cycles
DAY 7 (DAY 5 for high-throughput CUTseq)
12. Library size selection and purification
1. Transfer each sample into a new 1.5 ml LoBind tube
2. Add 70 μl (0.7x) of AMPure XP beads pre-warmed at room temperature
3. Mix thoroughly and incubate for 10 min at room temperature
4. Place the sample on a magnetic stand
5. Incubate for at least 5 min until the liquid appears clear
6. Carefully collect the supernatant and transfer it to a new 1.5 ml LoBind tube
7. Add 20 μl (0.2x) of AMPure XP beads pre-warmed at room temperature
8. Mix thoroughly and incubate for 5 min at room temperature
9. Place the sample on a magnetic stand
10. Remove and discard the supernatant
11. Wash the beads twice with 200 μl of freshly prepared 80% ice-cold ethanol
12. Air-dry the beads at room temperature
Note: do not dry the beads for more than 5–8 min, since this may result in low DNA yield
13. Remove the sample from the magnetic stand
14. Resuspend the beads in 10–20 μl of nuclease-free water
15. Incubate for 2 min at room temperature
16. Place the sample back on the magnetic stand
17. Incubate for at least 5 min until the liquid appears clear
18. Transfer the supernatant to a new 1.5 μl DNA LoBind tube
19. Check the library concentration using Qubit
20. Check the library quality and size on a Bioanalyzer using a high sensitivity DNA kit
21. Store the library @ –20 °C