Cell preparation
1. Wash the cells with 1×DPBS containing MgCl2 and CaCl2 pre-warmed to 37 °C and aspirate the DPBS from the flask
2. Add TrypLE™ Express Enzyme and incubate at 37 °C to dissociate cells
3. Add an equal volume of culture medium to neutralize the enzyme and resuspend the cells
4. Count cell number and take 1×106 cells
NOTE: For suspension cells, step 1-3 can be skipped
5. Pellet cells at 100-400g for 5 min (differs per cell type)
6. Wash cells with 1×PBS/5mM EDTA for once
7. Pellet cells at 100-400 g for 5 min
NOTE: For live cells, step 8-12 can be skipped for immediate sorting
8. Resuspend cells in 3 mL 1×PBS/5mM EDTA, incubate at RT for 1min
9. Add 3 mL of 8% PFA (in 1×PBS/5mM EDTA) and pipette up and down for few times
NOTE: Using 5% BSA pre-coated tubes can reduce cell loss caused by the fixation step
10. Incubate for 10 min in the dark
11. Add 315 μL of 2.5 M Glycine (final conc. 125 mM)
12. Pellet cells at 300 g for 5min
13. Resuspended cells in 1 mL 1xPBS/5mM EDTA (final density ~1x106/mL) and transfer the cell suspension to the BD FACS tubes
14. Keep the live/fixed cells on ice for immediate use, or store the fixed cells in 1xPBS/5mM EDTA/0.05% NaN3 at 4 °C for up to one month
Nuclei extraction from frozen tissue
1. Prepare the following stock solutions
● Sodium citrate tribasic dihydrate (for 1M): dissolve 14.70 g in 50 mL nuclease-free water
● Spermine tetrahydrochloride (for 0.5 M): dissolve 8.70 g in 50 mL nuclease-free water
● Tris (Hydroxymethyl) aminomethane (for 0.5 M): dissolve 3.03 g in 50 mL nuclease-free water
● IGEPAL CA-630 (for 10%): dilute 1 mL in 9 mL nuclease-free water
● HCl (for 1M): dilute 4.175 mL of 36% HCl in 45.825 mL nuclease-free water
NOTE: Store the above stock solutions at RT for up to 2 months
● Collagenase (for 8mg/mL): dissolve 8 mg in 1mL 1×PBS
● Dispase II (for 8mg/mL): dissolve 8 mg in 1mL 1×PBS
NOTE: Store the enzymatic stock solutions at -20° C for up to 6 months
2. Prepare the following working solutions
● Spermine Solution (100 mL, pH 7.6)
Sodium citrate tribasic dihydrate (1M) 340 μL
Spermine tetrahydrochloride (0.5M) 300 μL
Tris (Hydroxymethyl) aminomethane (0.5M) 100 μL
IGEPAL CA-630 (10%) 1 mL
Nuclease-free water 98.26 mL
NOTE: Adjust pH to 7.6 using 1M HCl solution. Store at 4 °C for up to 1 month
● Stop Solution (50 mL)
Trypsin inhibitor from chicken egg white, Type II-O 25 mg
Ribonuclease A from bovine pancreas, Type I-A 5 mg
Spermine solution (pH 7.6) 49.8 mL
NOTE: The trypsin inhibitor and ribonulease A powders are very light, be careful when handling these powders. Adjust pH to 7.6 using 1M HCl solution. Store at 4 °C for up to 1 month
● Nuclei Fixation Solution (20 mL)
Methanol 13.3 mL
Acetic Acid 6.6 mL
NOTE: Prepare the nuclei fixation solution in glassware. Store at -20 °C
3. Before each processing, incubate Spermine Solution and Stop Solution at room temperature for at least 10 min. Thaw the enzymatic stock solutions and place them on ice
4. Prepare a dry ice bucket
5. For each sample to be processed: pre-chill on dry ice a pair of sterile scalpels and one sterile Petri dish.
6. Prepare fresh Tissue Lysis Solution before each use (volumes for one sample)
Trypsin-EDTA (0.25%), phenol red 24 μL
Collagenase (8mg/mL) 25 μL
Dispase II (8mg/mL) 25 μL
Spermine Solution (pH 7.6) 1.926 mL
7. Retrieve cryovial containing the tissue fragment(s) from -80 °C freezer and always keep on dry ice
8. Transfer the fragment of tissue into the pre-chilled Petri dish placed on top of the dry ice.
9. From the 2 mL of tissue lysis solution, pipette 200 μL on top of the tissue fragment
10. Incubate for 2-3 minutes until the tissue lysis solution on top of the tissue fragments freezes
NOTE: The freezing step in tissue lysis solution provides the tissue with a more homogeneous density and increases the efficiency of tissue mincing, enzymatic disaggregation, and nuclei recovery
11. Using a chilled disposable sterile scalpel, mince the tissue fragment(s) embedded in Tissue Lysis Solution thoroughly
12. Remove the Petri dish (containing the minced tissue and Tissue Lysis Solution) from dry ice and bring to RT
13. Continue mincing the tissue until the Tissue Lysis Solution is completely thawed and the tissue fragments can flow through a 1 mL tip without clogging
14. Use the remaining 1800 μL of Tissue Lysis Solution to rinse and transfer all the tissue fragments from the Petri dish back to a 5 mL LoBind tube
15. Place the tube on a sample mixer and incubate at RT for 15 minutes, rotating at 20 rpm (or very low speed). Check the tissue digestion every 5 min
NOTE: The tissue that is not successfully digested in 15 minutes, likely will not digest with longer incubations
16. Add 2 mL of Stop Solution. Mix by inverting the tube gently
17. Wash the 50 μm cell strainer with 1 mL of spermine solution
18. Filter the nuclei suspension through the 50 μm cell strainer and collect the flow-through in the 5 mL LoBind tube
19. Centrifuge the flow-through containing the nuclei at 300 g for 5 min at RT
NOTE: Increasing centrifugation speed or time will result in increased nuclei clumping and lead to excess amounts of cellular debris.
20. Carefully discard the supernatant and resuspend the nuclei in 400 μl of Nuclei Fixation Solution, by pipetting up and down
NOTE: For live nuclei, the nuclei fixation step can be skipped for immediate sorting
21. Incubate the nuclei suspension on ice for 15 min
22. Centrifuge the nuclei suspension at 300 g for 5 min at RT
23. Carefully discard the supernatant and resuspend the nuclei in 1 mL 1×PBS/5mM EDTA
24. Filter the nuclei suspension through either 10 or 20 μm cell strainer to the BD FACS tubes (test which best fit for the nuclei size of each tissue)
25. Keep the nuclei suspension on ice for immediate use, or store the fixed nuclei suspension in 1xPBS/5mM EDTA/0.05% NaN3 at 4 °C for up to one month
Single cell / nuclei sorting
NOTE: No matter which FACS machine is used, always set the nozzle size at 100 μm and make sure the FACS droplet volume is below 10nL. The excessive volume will dilute the downstream reactions and the excessive EDTA will inhibit the following enzymatic activity. The 384-well plates are better suited when dispensing with nanoliter volumes as the conical bottom and size of the wells allow easier visualization of the dispensed droplets inside the Vapor-Lock (oil phase).
For live / fixed cells
1. Dilute the Hoechst stock solution (12.3mg/ul) 1:10 with water. Add 2 μL diluted Hoechst solution to each 1mL of cell suspension (final conc. 2.46ng/ul) and mix thoroughly
2. Incubate for at least 30-40min at 37°C while shaking at 600rpm in the dark
3. After staining, the tubes can be kept on ice in the dark before sorting (continue with step4)
For live / fixed nuclei
1. Dilute the Hoechst stock solution (12.3mg/ul) 1:10 with water. Add 2 μL diluted Hoechst solution to each 1mL of nuclei suspension (final conc. 2.46ng/ul) and mix gently and thoroughly
2. Incubate for 2 min on ice in the dark
3. After staining, the tubes can be kept on ice in the dark before sorting (we recommend at +4 °C or on ice)
4. Aliquot 5μL of Vapor-Lock per well with the multiple-channel pipette in the targeted region of 384-well plates
5. Bring the FACS tubes, Vapor-Lock prefilled plates and the adhesive films to the FACS machine
6. When sorting, set 10nL as the FACS droplet volume and 100 μm as the FACS nozzle size
7. Leave empty wells randomly as positive controls or negative controls
8. After sorting of each plate, seal the 384-well plate immediately with adhesive film and spin down at 300 g for 5min. Store the plates at 4 °C for intermediate use, or place the plates at -20 °C for long term storage
Positive and Negative Controls Dispense
1. Incubate the 384-well plate at 4 °C for 10min if the plates are stored at -20 °C
2. Serially dilute the corresponding extracted bulk genomic DNA (gDNA) to 2ng/μL as the positive control. Use fixed gDNA for fixed cells, while using non-fixed gDNA for live cells
3. Set up the program on I.DOT and dispense positive controls and negative controls at the same time.
● For positive controls, dispense 10nL of 2ng/μL gDNA (20pg gDNA per well)
● For negative controls, dispense 10nL of 1×PBS/5mM EDTA
MALBAC Cell Lysis
NOTE: All the following volumes for MALBAC are prepared for a single well in a 384-well plate, and we recommend preparing for at least 48 wells each time, since it will be hard to prepare the reaction mix with a pipette.
1. Prepare the Cell Lysis Reaction Mix by combining the following components and mix well by pipette up and down before adding to the source well in I.DOT
Cell Lysis Buffer 25 nL
Cell Lysis Enzyme 0.5 nL
2. Dispense 25.5 nL per well. Shake and centrifuge the plate
NOTE: From now on, after dispensing for each step, we shake the plate in a ThermoMixer at 1,000 rpm for 1 min and centrifuge at 3,220 g for 5 min before each incubation.
3. Place the plate in QuantStudioTM 5 with lid set at 80°C following the program below
i. 50°C 2 h
ii. 80 °C 10 min
iii. 4°C forever
NOTE: Lysis at 50°C can be reduced to 1h, and we observed no difference. When handling many plates together, we recommend using an incubator instead of a thermocycler. Proceed immediately to the next step or place the plate at -20°C for long term storage
MALBAC Pre-Amplification
1. Prepare the Pre-Amplification Reaction Mix by combining the following components and mix well before adding to the source well in I.DOT
Pre-Amplification Buffer 150 nL
Pre-Amplification Enzyme Mix 5 nL
2. Dispense 155nL per well. Shake and centrifuge the plate
3. Place the plate in QuantStudioTM 5 following the program below.
i. 94 °C 3 min
ii. 20 °C 40 sec
30 °C 40 sec
40 °C 30 sec
50 °C 30 sec
60 °C 30 sec
70 °C 4 min
95 °C 20 sec
58°C 10 sec
GO TO step ii, 8 times
iii. 4 °C forever
NOTE: After this step, proceed immediately to the next step
MALBAC Exponential Amplification
1. Prepare the Amplification Reaction Mix by combining the following components and mix well before adding to the source well in I.DOT
Amplification Buffer 150 nL
Amplification Enzyme Mix 4 nL
SYBR Green I (4x) 11 nL
2. Dispense 165 nL per well. Shake and centrifuge the plate
3. Place the plate in QuantStudioTM 5 following the program below
i. 94 °C 30 sec
ii. 94 °C 20 sec
58 °C 30 sec
72 °C 3 min
GO TO step ii, 16 times
iii. 4°C forever
NOTE: Proceed immediately to next step or place the plate at -20°C for long term storage. Sample amplification efficiency can be monitored using a real-time thermal cycler by adding SYBR Green I dye at 0.125 × final concentration in the Amplification Reaction Mix. Data analysis should be performed on raw background-subtracted (not baseline cycle normalized) fluorescence, and the instrument/software should be set to the appropriate mode. It is not necessary to monitor the MALBAC amplification efficiency of each plate, one can simply use the 384-well PCR thermocycler.
DNA Digestion
1. Prepare the Digestion Mix by combining the following components and mix well before adding to the source well in I.DOT
CutSmart Buffer (10×) 50 nL
NlaIII-HF (10,000U/ml) 100 nL
2. Dispense 150 nL per well. Shake and centrifuge the plate
3. Place the plates in QuantStudioTM 5 following the program below
i. 37 °C 1 h
ii. 65 °C 20 min
iii. 4 °C forever
NOTE: When handling many plates together, we recommend using an incubator instead of a thermocycler. After this step, proceed immediately to next step
Ligation of scCUTseq adapters
1. Dispense 300 nL of 33 nM scCUTseq adapter per well
NOTE: In order to be pooled together, different wells must use unique barcodes
2. Prepare the Ligation Mix by combining the following components and mix well before adding to the source well in I.DOT
For Rapid Ligase Reaction
Nuclease-free water 50 nL
T4 Rapid Ligase Buffer (5×) 300 nL
ATP (10 mM) 120 nL
BSA (50 mg/ml) 30 nL
T4 Rapid Ligase 200 nL
Alternatively, when using standard ligase:
For Standard Ligase Reaction
Nuclease-free water 200 nL
T4 Ligase Buffer (10×) 150 nL
ATP (10 mM) 120 nL
BSA (50 mg/ml) 30 nL
T4 Standard Ligase 200 nL
3. Dispense 700 nL per well. Shake and centrifuge the plate
4. Incubate at 22 °C for 30 min for rapid ligase, and 1h for standard ligase.
5. Add 5 μL EDTA solution (33 mM in Nuclease-free water for a final concentration of 25mM) to each well with a multiple-channel pipette to stop the ligation reaction as well as to bring up the volume
NOTE: Adding 5ul EDTA solution before pooling is important. It will help to increase the recovery as well as to stop the mis-ligation between different adapters during pooling.
6. Pool the contents of all the wells by placing the plate upside down on a Nunc Rectangular Dish covered with parafilm, seal the aligned skirts with tape and centrifuge at 120 g for 1 min
7. Collect all the liquid in the dish to one 5 mL DNA LoBind tube
8. Briefly centrifuge the tube and carefully discard the upper oil phase of the liquid (containing Vapor-Lock)
9. Measure the volume of the lower aqueous phase with a pipette
NOTE: After this step, proceed immediately to the next step
DNA cleanup
1. Mix with 1.2× vol/vol ratio of AMPure XP beads pre-warmed at RT
2. Mix thoroughly and incubate for 10 min at RT
3. Place the sample on a magnetic stand
4. Incubate for at least 10 min until the liquid appears clear
5. Remove and discard the supernatant
6. Wash the beads twice with freshly prepared 80% ethanol (the volume should be high enough to cover the beads)
7. Air-dry the beads at RT for 5-10 min
NOTE: Do not over-dry the beads, since this may result in low DNA yield
8. Remove the sample from the magnetic stand
9. Resuspend the beads in 200 μL of Nuclease-free water
10. Incubate for 2 min at RT
11. Place the sample back on the magnetic stand
12. Incubate for at least 5 min until the liquid appears clear
13. Transfer 190 μL of supernatant to a new 1.5 mL DNA LoBind tube
14. Check the DNA concentration with 1 μL using Qubit DNA HS kit
NOTE: Proceed immediately to the next step or place the samples at -20 °C for long term storage
Sonication
1. Take 250ng of DNA from the above sample and add Nuclease-free water to bring up the volume to 55 μL
NOTE: Do not overload for this step. Usually, each Covaris tube can shear no more than 500ng of DNA
2. Transfer the DNA to the Covaris tubes and operate following the Covaris manual
NOTE: To sonicate DNA, we typically use a Covaris ME220 Focused-ultrasonicator with microTUBE-50 AFA Fiber Screw-Cap tubes or strips, with a target peak of 200bp. Note that we sonicate DNA independently of the restriction enzyme used, since we have found that this results in higher-quality libraries
SpeedVac
1. Transfer sample from Covaris tube to a new 0.5 mL DNA LoBind tube
2. Proceed to SpeedVac Vaccum Concentrator with a medium heating mode until the volume of the sample reaches 8 μL
In vitro transcription (IVT)
1. Mix the following reagents on ice, and add 12 μL each sample
rATP+rUTP+rGTP+rCTP 8 μL
T7 Polymerase Buffer (10×) 2 μL
T7 Polymerase 1.5 μL
RNaseOUTTM Recombinant Ribonuclease Inhibitor 0.5 μL
NOTE: Prepare from separate rNTP solutions provided with MEGAscript® T7 Transcription Kit
2. Incubate for 14 hours at 37 °C in a PCR thermocycler with the lid set at 70 °C
NOTE: Instead of overnight incubation, 2h of incubation also works well. After this step, proceed immediately to the next step
RNA cleanup
1. Add 1 μL of DNase I (RNase-free) to the IVT product
2. Incubate for 15 min at 37 °C
3. Bring up the volume to 50 μL by adding 29 μL Nuclease-free water, then mix with 90 μl (1.8x vol/vol) of RNAClean XP beads pre-warmed at RT
4. Mix thoroughly and incubate for 10 min at RT
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% ethanol
9. Air-dry the beads at RT
NOTE: Do not over-dry the beads, since this may result in low RNA yield
10. Remove the sample from the magnetic stand
11. Resuspend the beads in 10 μL of Nuclease-free water
12. Incubate for 2 min at RT
13. Place the sample back on the magnetic stand
14. Incubate for at least 5 min until the liquid appears clear
15. Transfer 8.8 μL of supernatant to a new 0.5 mL DNA LoBind tube
16. Check the RNA concentration with 1 μL using Qubit-BR RNA kit
NOTE: After this step, proceed immediately to the next step
RA3 adapter ligation
1. Add 1 μL of 10 μM RA3 adapter to 7.8 μL obtained after RNA cleanup
2. Incubate for 2 min at 70 °C in a PCR thermocycler, then immediately place the sample on ice
3. Add 3.2 μL of the following mix
RNA Ligase Buffer (10×) 1.2 μL
RNaseOUTTM Recombinant Ribonuclease Inhibitor 1 μL
T4 RNA Ligase, truncated 1 μL
4. Incubate for 2 hours at 25 °C in a PCR thermocycler with the lid set at 30 °C
NOTE: After this step, proceed immediately to the next step
Reverse transcription (RT)
1. Add 2 μL of 10 μM RTP primer
2. Incubate for 2 min at 70 °C in a PCR thermocycler, then immediately place the sample on ice
3. Add 11 μL of the following mix
SSIV Buffer (5×) 5 μL
dNTPs (12.5 mM) 1 μL
DTT (0.1M) 2 μL
RNaseOUTTM Recombinant Ribonuclease Inhibitor 1 μL
SSIV Reverse Transcriptase 2 μL
4. Incubate the samples in a thermos cycler follow the program below
i. 50 °C 20 min
ii. 65 °C 10 min
iii. 4 °C forever
NOTE: Proceed immediately to the next step or place the samples at -20°C for long term storage
Library indexing and amplification
1. Add 8 μL per sample of the desired indexed Illumina primer (10μM)
2. Add 167 μL of the following mix
Nuclease-free water 59 μl
NEBNext® UltraTM II PCR Master Mix 100 μl
RP1 primer (10 μM) 8 μL
3. Divide the final mix of each sample into 4 PCR tubes with each containing 50 μL
4. In a PCR thermocycler perform the following program
i. 98 °C 30 sec
ii. 98 °C 10 sec
60 °C 30 sec
65 °C 45 sec
GOTO step ii, 10 times
iii. 65 °C 5 min
iv. 4 °C forever
NOTE: Adjust PCR cycles accordingly to the input to the IVT step. Proceed immediately to the next step or place the samples at -20°C for long term storage
Library cleanup
1. Pool the 8 tubes for each sample in a new 1.5 mL LoBind tube
2. Add 0.8× vol/vol ratio of AMPure XP beads pre-warmed at RT
3. Mix thoroughly and incubate for 10 min at RT
4. Place the sample on a magnetic stand
5. Incubate for at least 5 min until the liquid appears clear
6. Remove and discard the supernatant
7. Wash the beads twice with 1 mL of freshly prepared 80% ethanol
8. Air-dry the beads at RT
NOTE: Do not over-dry the beads, since this may result in low DNA yield
9. Remove the sample from the magnetic stand
10. Resuspend the beads in 50 μL of nuclease-free water
11. Incubate for 2 min at RT
12. Place the sample back on the magnetic stand
13. Incubate for at least 5 min until the liquid appears clear
14. Transfer the supernatant to a new 1.5 mL DNA LoBind tube
15. Check the library concentration using Qubit dsDNA HS kit
16. Check the fragment distribution on a Bioanalyzer 2100 using DNA HS chip
NOTE: Place the libraries at -20 °C for long term storage