Main reference is (Martin et al., 2023). All modifications/improvements are derivative of this base protocol.

Note that their volume estimate is way overkill, 10% extra for pipetting is enough.

Changes are in bold.

To print with the sidebar, resize your browser window to be narrow until the sidebar merges with the main text.

Table of Contents

• Reverse Transcription
• Ligation
• Final distribution
• Second-strand synthesis
• Protease Digestion
• Tagmentation
• Library Amplification
  • Concentration of library
• References

Protocol

Dephosphorylation

1. After sonification, centrifuge sample.
2. Aspirate sample to nuclei pellet and add 50 μl SPBSTM. Place sample in a PCR tube.
3. Add 1:40 SAP enzyme.
4. Incubate sample at 30C for 30 min.
5. Stop incubation, add 50 μl SPBSTM + 30 mM EDTA. Mix.
6. Incubate the sample at 65°C for 5 minutes.
7. Add 3 μl of 1M MgCl2.
8. Wash twice using a swinging bucket centrifuge.

Reverse Transcription

The target is 2.4M cells/plate or 200,000 cells/column.

1. Resuspend an aliquot of frozen nuclei in 500 μl of 0.3 M SPBSTM to start and count. Dilute nuclei if necessary to get an accurate count. If nuclei are clumpy, sonicate (Diagenode Bioruptor Plus) on low intensity for 12 s only at 4°C. If there is still an excess of clumps, put the nuclei over a 40-μm Flow-mi pipette tip filter before counting. Use of a Flow-mi filter is a last resort because it results in nuclei loss but is helpful if you have an excess of nuclei.

2. Bring predispensed primer plates out and quickly spin. There’s 2 μl of primer in each well.

3. Set the thermal cycler up to be ready (lid at 65°C). (from ISAACC-Seq) | Step | Temperature | Time | | ---- | ------------------------------------ | ------ | | 1 | 50°C | 10 min | | 2 | 8°C | 12  s | | 3 | 15°C | 45  s | | 4 | 20°C | 45  s | | 5 | 30°C | 30  s | | 6 | 42°C | 2  min | | 7 | 50°C | 3  min | | 8 | 65°C | 1 min | | | Go to step 3, 2 more times (3 total) | | | 9 | 50 °C | 5 min |

4. Setup the master mix. PEG is viscous and is slow to mix with water. Make sure that everything well mixed before proceeding. Do NOT use 5x Maxima buffer as it contains DTT (unless you used DSS then it’s fine.) | RT mix per plate | 1 well | x100 | | --------------------------------- | ------ | ---- | | 5× Superscript IV buffer (μl) | 2 | 200 | | dNTPs | 0.5 | 50 | | 50% PEG | 1.5 | 150 | | Maxima RT H Minus (μl) | 0.25 | 25 | | RNaseIn Plus | 0.05 | 5 | | Nuclease-free H₂O (μl) | 3.75 | 375 |

5. Pull out the desired amount of nuclei into a new tube and spin. Remove the supernatant and resuspend nuclei in the RT reaction mix.

6. Dispense 8 μl of reaction mix into each well (65 μl of mix × 8 in a strip tube for multichannel pipetting). Do not touch the bottom of the plate if you do not want to change tips.

7. Start the thermal cycler program.

8. Ice plates until they are cold (>2 min).

9. Add 9 μl of cold SPBSTM per well. _To maximize recovery, pool wells by using a 12-multichannel pipette with 200 μl tips to pipette gently up and down (the pipetting up and down is important to dislodge the nuclei, but try to avoid creating excessive bubbles) and combine each row of the plate into the bottom row. You can use the same tips for the whole plate. Collect these wells into two cold microcentrifuge tubes. (The solution will be bubbly, so it is difficult to squeeze into one tube, although if you do it properly, one tube is enough (or use 2 ml tubes))

10. Spin at 1,000 g for 3 min at 4°C. The pellet will be small, but you should be able to see it. Remove and discard the supernatant.

11. Combine tubes and wash once more in 1 ml of cold 0.3 M SPBSTM. Spin at 1,000 g for 3 min at 4°C. Remove and discard the supernatant.

Ligation

1. Thaw and spin down the ligation primer plate.
2. Prepare the ligation mix on ice:

3. Resuspend nuclei in the ligation mix at 4°C
4. Distribute 8 μl to each well of a new plate on ice.
5. Re-cover the plate with seals, spin-down, vortex at 2,000 rpm for 10 s, and spin-down.
6. Incubate for 10 min at room temperature.
7. Ice plates until cold.
8. Add 10 μl of cold SPBSTM to each well. This helps keep the nuclei from clumping and allows for more nuclei to be recovered.
9. Pool wells by using a 12-multichannel to pipette gently up and down (the pipetting up and down is important to dislodge the nuclei) and combine each row of the plate into the bottom row. Then, collect these wells into two cold microcentrifuge tubes.
10. Spin at 1,000 g for 3 min at 4°C. Remove and discard the supernatant.
11. Combine the two tubes and wash once more with 1 ml of 0.3 M SPBSTM per wash.

Final distribution

1. Spin down and resuspend in PBS with 0.5% DNase-free BSA w/ 1:1000 RNaseIn and 0.1 μg/ml DAPI for sorting. Do not use SPBSTM, the nozzle will leak due to the presence of Triton.
2. Sort nuclei into a 1.5 ml tube coated with 100 μl SPBSTM.
3. Centrifuge at 1,000 g for 3 min at 4°C. Resuspend in 1× second-strand buffer + 0.1% Triton X-100 to prevent clumping, targeting 4 μl/well. Base is 1,000 cells/well with 1 ligation plate, increase the concentration as needed.
4. If proceeding, add the enzyme mix. We usually sort an extra plate with a few extra strip of tubes for diagnostics.
5. Cover with seals and freeze plates at -80°C or proceed with second-strand synthesis.

Second-strand synthesis

1. Thaw plate on ice.
2. Make second-strand synthesis mix on ice as follows:

3. Put 1 μl of second-strand synthesis mix into each well (17 μl of mix ×8 in a strip tube for multichannel pipetting).
   The total volume per well is now 5 μl.
4. Incubate for 2.5 h at 16°C in a PCR machine (no heated lid).

Protease Digestion

1. Add 1 μl of protease to each well.

2. Incubate at 37°C for 1 h and heat-inactivate the protease at 75°C for 20 min in a PCR machine (80°C heated lid).

Tagmentation

1. On ice, make tagmentation mix as follows: (the extra 25 μl is extra volume.)

   Reaction mix per plate	1well	x100
   2x Tagment DNA buffer (μl)	2.5	250
   Nuclease-free H2O (μl)	2.5	250
   N7-loaded Tn5 (μl)	0.125	12.5

2. To an 8-strip tube, add 62 μl to each well. On ice, dispense 5 μl to each well. The total volume per well is now ~10-11 μl.

3. Incubate for 5 min at 55°C in a PCR machine, heat-inactivate the transposase at 80°C for 5 min (85°C heated lid) and ramp down to 4°C.

4. Step	Temperature	Time
   1	55°C	7 min
   2	80°C	5 min
   3	4°C	15 s

Library Amplification

1. Prepare the primer plate 4 μl of indexed P7 primers (10 μM) to each well.

2. Add 20 μl of 2× Takara Ex Premier PCR mix and 6 μl of nuclease-free water to each well. You can make a master mix for this. Vortex at 2,000 rpm for 10 s and spin down.

3. Amplify in a thermocycler by using 17-19 cycles withOUT a pre-extension step in the following program: | Step | Temperature | Time | | ---- | --------------------------- | ----- | | 1 | 94°C | 1 min | | 2 | 98°C | 10 s | | 3 | 63°C | 15 s | | 4 | 68°C | 45 s | | 5 | go to Step 3, 16-18 more times | | | 6 | 68°C | 3 min | | 7 | 4°C | ∞ |

4. Pick a few wells, dilute the product 20-fold in DI and run on a Tapestation D5000 HS assay to check for amplification.

Concentration of library and agarose gel purification

1. Pool 5 μl of each well and do a 0.8× AMPure XP cleanup (230 μl of beads) by following the manufacturer’s protocol.
   Save the remaining plate by covering with foil and storing at room temperature or at 4°C for ≤6 months, in case you need to redo cleanup or if you anticipate needing more library for a large NovaSeq run.
2. Wash the AMPure bead pellet twice gently with 70% (vol/vol) ethanol and elute the pool in 50 μl.
3. Perform size selection with 0.55-0.8x SPRISelect beads or homemade beads. See 10x v3 protocol for details. Or gel extraction for maximum recovery
4. Run the library on NextSeq (or NovaSeq depending on the final cell numbers or sequencing depth desired) by using standard primers. (36x10x10xremaining)

References