Tissue Fixation
1. Place mortar and pestle, 15 mL conical tube, and a metal spatula in dry ice for 15 minutes. (Do this in a styrofoam box with lid on to prevent condensation).
2. Pulverize the brain sample using the mortar and pestle.
3. Scrape the powdered tissue using a metal spatula into a 15 mL conical tube and resuspend the tissue in 5 mL of PBS.
4. Add 276 µL of 37% formaldehyde and incubate at room temperature for 10 minutes with slow rotation.
5. Add 447 µL of 2.5 M glycine solution to quench the reaction. Incubate at room temperature for 5 minutes with slow rotation.
6. Centrifuge for 10 minutes at 1000 rpm at 4°C. Discard supernatant into an appropriate collection container.
7. Resuspend cells in 1mL of PBS and transfer the sample to 1.75 mL eppendorf tube. Spin for 5 minutes at 1000 rpm at 4°C. Discard supernatant and flash-freeze cell pellets in liquid nitrogen or dry ice.
8. Either proceed to the rest of the protocol or store cell pellets at -80°C.
Lysis and Restriction Digest
Lysis Buffer:
50µL 1M CaCl2
30µl 1M MgAc
40µl 0.5M EDTA
10µl 0.5M EGTA
100 µl 1M Tris-HCl pH 8
400 µl 50X stock protease inhibitors (Roche)
10 µl 1M DTT
10 µl 0.1M/ml PMSF
9.35 mL Water
*Dissolve one tablet - Complete protease inhibitor in 1mL water to get 50X stock.
* Add 400 µl of 10% Triton-X 100 to 8 mL of Lysis buffer to make Lysis buffer with 0.4% Triton-X.
9. Prior to starting the protocol, place a M-tube (Miltenyi Biotec 130-096-335) and a 50 mL conical tube in ice for 15 minutes. Prepare lysis buffer without and with 0.4% Triton-X 100 (Sigma 93443) and place it in ice. (For processing one crosslinked pellet, prepare 5 mL of each of Lysis buffer without and with 0.4% Triton-X 100).
10. Add 1 mL of ice-cold lysis buffer to one crosslinked pellet of cells and resuspend the cells. Transfer the sample to a M-Tube.
11. Add another 2 mL of the lysis buffer to the M-tube. Insert the M-tube to gentleMACS Dissociator and run the gentleMACS factory-set program: “Protein-M-tube-1.0”.
12. Transfer the M-tube to ice. Pour the sample through a 40 µm nylon cell strainer (Corning 431750) over the 50 mL conical tube kept in ice. Add 3 mL of Lysis buffer with 0.4% Triton-X to the M-Tube. Close the M-tube with its cap and invert gently a couple of times to wash the tube and pour it over the cell strainer.
13. Pool both the lysis buffer together (one without Triton-X and the other with 0.4% Triton-X) to make a 0.2% Triton-X solution.
14. Wash the cell strainer with 2 mL of Lysis buffer with 0.2% Triton-X. Discard the cell strainer.
15. Centrifuge the 50 mL conical tube at 1000 rpm for 10 minutes at 4 C. Discard the supernatant carefully. (Remove the supernatant carefully using vacuum. Do not invert the tube to discard the supernatant).
16. Resuspend the pellet in 1 mL of Lysis buffer with 0.2% Triton-X and transfer it to a 1.75 mL Eppendorf tube.
17. Centrifuge at 2500 G for 10 minutes at 4 C. Discard the supernatant.
18. Gently resuspend pellet in 50 µl of 0.5% sodium dodecyl sulfate (SDS) and incubate at 62°C for 10 minutes, shaking.
19. After heating is over, add 145µl of H2O and 25µl of 10% Triton X-100 to quench the SDS. Mix well, avoiding excessive foaming. Incubate at 37°C for 15 minutes, shaking.
20. Add 25µl of 10X NEB Buffer 4 (NEB B7004S). Then add 20 µl (200U) of NlaIII restriction enzyme (NEB R0125) and digest chromatin ON at 37°C, shaking.
Proximity Ligation and Staining
21. Incubate at 62°C (shaking) for 20 minutes to inactivate NlaIII, then cool to room temperature.
22. Add 900µl of ligation master mix to sample
Ligation Buffer:
* 663µl H2O
* 120µl 10X NEB T4 DNA ligase buffer (NEB B0202)
* 100µl 10% Triton X-100
* 12µl 10mg/mL Bovine Serum Albumin
* 5µl 400U/µl T4 DNA Ligase (NEB M0202)
23. Mix by inverting and incubate at room temperature for 4 hours with slow rotation.
24. Add 15 µl of 0.1 µg/µl of Hoechst (Life Technologies H3570) for the last 30 minutes of the ligation step.
25. Spin the sample at 2500 G for 5 minutes and discard the supernatant. Resuspend the pellet in 600 µl of PBS. Add 6 µl of 0.1 µg/µl of Hoechst to the sample.
26. Pass the sample through a 40 µm nylon cell strainer (Corning 431750) into another 1.75 mL eppendorf tube.
27. Centrifuge sample for 5 minutes at 2500g, aspirate supernatant carefully
28. (Optional) If using cellular markers for FACS: Resuspend pellet in 900µL of DPBS + RNase inhibitors
29. (Optional) If using cellular markers for FACS: Add 100µL of UltraPure BSA (50mg/mL)
30. (Optional) If using cellular markers for FACS: Incubate on ice for 20 minutes
31. (Optional) If using cellular markers for FACS: add appropriate antibodies. For example, to enrich for neural populations from human brain samples, we add 1µL of AlexaFluor 488 conjugated anti-NeuN antibody, incubate on ice for 20 minutes.
32. (Optional) If using cellular markers for FACS: Centrifuge sample for 5 minutes at 2500g, aspirate supernatant carefully
33. Reuspsent pellet in 1mL of DPBS + RNase inhibitors
Cell Sorting
34. Prepare 384 well plates for cell sorting as described in “Reagents Setup” of “Reagents” section.
35. Sort cells using FACS into 384 well plates. Nuclei are first gated based on Forward Scatter (FSC) and Side Scatter (SSC) pulse height, then multiplet exclusion gating was applied (forward scatter and side scatter pulse width). Finally, nuclei of specific DNA content were selected (e.g. 2N) by virtue of Hoechst fluorescence intensity.
36. After sorting, plates are incubated at 50?C for 20 minutes in a 384 well PCR block with heated lid for proteinase K digestion.
37. At this point, plates can be sorted at -20?C until ready for further processing.
Bisulfite Conversion
Timing ~ 5h
38. Add 15µL CT conversion reagent to each well of 384-well plate. Pipette up and down for 8 times to mix the sample.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - 15uL_Conversion_Buffer
39. Seal the plate with adhesive film and quick spin for 10s at 2,000xg at room temperature. Place the plate in a thermocycler and run the following program:
98°C 8 min
64°C 3.5hrs
4°C Hold
40. Load 80µL M-Binding buffer to each well of 384-Well DNA Binding Plate.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - 80uL_Binding Buffer
41. Transfer bisulfite conversion reactions to Zymo-Spin 384 Well DNA Binding Plate. Pipette up and down for 8 times to mix the sample. Place the 384-Well DNA Binding Plate on a 2.0mL 96-well Deep Well Plate and centrifuge for 5 min at 5,000g. Discard the flow through by decanting.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - 17uL_Conversion_Rxn_To_Binding_Buffer.
42. Add 100µL M-Wash Buffer to each well of 384-Well DNA Binding Plate. Centrifuge for 5 min at 5,000g and discard the flow through by decanting.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - 100uL_M_Wash_Buffer.
43. Add 50µL M-Desulphonation Buffer to each well of 384-Well DNA Binding Plate. Incubate at room temperature for 15 min. Centrifuge for 5 min at 5,000g and discard the flow through by decanting.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - 50uL_Desulphonation_Buffer.
44. Add 100µL M-Wash Buffer to each well of 384-Well DNA Binding Plate. Centrifuge for 5 min at 5,000g and discard the flow through by decanting.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - 100uL_M_Wash_Buffer.
45. Repeat step 7.
46. Place 384-Well DNA Binding Plate on new 384-well PCR plate. Add 7µL Random Primer Solution. Each quadrant of 384-well plate is barcoded with a distinct indexed random primer (Fig. 2). Every two 384-well plates receive a complete set of all eight indexed random primers. Incubate for 5 min at room temperature. Centrifuge for 5 min at 5,000g and discard the 384-Well DNA Binding Plate.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - 7uL_Primer_Elution.
47. Seal the plate with adhesive film and store at -20°C for up to 1 week.
Random-primed DNA synthesis
Timing ~2h
48. Denature the samples by placing 384-well PCR plate on a thermocycler and run the following program.
95°C 3 min
Immediately place the plate on ice for 2 minutes.
49. Add 5µL Random Priming Master Mix to each well of the 384-well PCR plate. Vortex and quick spin for 10s at 2,000xg.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - 5uL Random_Priming_Mix.
50. Place the plate in a thermocycler and run the following program:
4°C 5 min
25°C 5 min
37°C 60 min
4°C Hold
Inactivation of free primers & dNTP
51. Add 1.5µL Exo/rSAP Master Mix to each well of the 384-well PCR plate. Vortex to mix the samples and quick spin for 10s at 2,000xg.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - 1uL_Exo_rSAP_MM.
52. Place the plate in a thermocycler and run the following program
37°C 30 min
4°C Hold
Sample clean-up
53. Add 73.6µL (0.8x) SPRI Beads to each well of four clean 96-well PCR plates.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - BP_Add_To_Clean_96well.
54. Transfer samples from two 384-well plates to each 96-well plate. The eight quadrants (from two 384-well plates) barcoded with distinct indexed random primers are combined. Mix the samples by vortexing and incubate for 5 minutes at room temperature, then quick spin for 10s at 2,000xg.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - BP_Transfer_Lib_384well_To_96well.
55. Place 96-well plates on DynaMag™-96 Side Magnet, let stand until solution in wells is clear of beads (~5 minutes). Remove supernatant and wash beads 3x with 150µL fresh 80% EtOH. Remove all EtOH, remove plates from magnet, and let beads dry at room temperature. DO NOT overdry beads.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - BP_2Plate_150uL_EtOH_Wash.
56. Add 10µL EB buffer and resuspend beads by pipet. Vortex and incubate for 5 minutes at room temperature, then quick spin for 10s at 2,000xg. Place back on magnet and let stand until solution is clear of beads (~5 minutes).
57. Remove 10uL supernatant to a clean 96-well PCR plate.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - 10uL_96Well_Elution.
Adaptase reaction
58. Denature the samples by placing 96-well plates on a thermocycler and run the following program.
95°C 3 min
Immediately place the plate on ice for 2 minutes.
59. Add 10.5µL Adaptase Master Mix to each well of the 96-well PCR plate. Vortex and quick spin for 10s at 2,000xg.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - 10uL_25uL_Adaptase_KAPA. Enter 10.5uL for the volume transferred.
60. Place the plate in a thermocycler and run the following program:
37°C 30 min
95°C 2 min
4°C Hold
Library amplification
61. Add 5uL PCR Primer Mix.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - 5uL_PCR_Primer.
62. Add 25µL 2x KAPA HiFi Mix. Vortex and quick spin for 10s at 2,000xg.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - 10uL_25uL_Adaptase_KAPA. Enter 25uL for the volume transferred.
63. Place the plate in a thermocycler and run the following program:
a. 95°C 2 min
b. 98°C 30s
c. 98°C 15s
d. 64°C 30s
e. 72°C 2 min
Go to step c 14 times
f. 72°C 5 min
g. 4°C Hold
Library clean-up
64. Sample cleanup. Add 40µL (0.8x) SPRI Beads to each well of four 96-well PCR plates. Transfer contents of one 96-well plate to another to combine four 96-well plates to two 96-well plates. Vortex and incubate for 5 minutes at room temperature, then quick spin for 10s at 2,000xg.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - BP_Add_Beads_And_Combine_96well.
65. Place 96-well plates on DynaMag™-96 Side Magnet, let stand until solution in wells is clear of beads (~5 minutes). Remove supernatant and wash beads 2x with 150µL fresh 80% EtOH. Remove all EtOH, remove plate from magnet and let beads dry at room temperature. DO NOT overdry beads.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - BP_2Plate_150uL_EtOH_Wash (stop script after two wash steps).
66. Add 25µL EB and resuspend beads by pipet. Vortex and incubate for 5 minutes at room temperature, then quick spin for 10s at 2,000xg. Place back on magnet and let stand until solution is clear of beads (~5 minutes). Remove 25uL supernatant to a clean 96-well PCR plate.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - 20uL_96Well_Elution.
67. Add 20µL (0.8x) SPRI Beads to each well of two 96-well PCR plates. Transfer contents of one 96-well plate to another to combine two 96-well plates to one 96-well plate. Vortex and incubate for 5 minutes at room temperature, then quick spin for 10s at 2,000xg.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - BP_Add_Beads_And_Combine_96well.
68. Place 96-well plate on DynaMag™-96 Side Magnet, let stand until solution in wells is clear of beads (~5 minutes). Remove supernatant and wash beads 2x with 150µL fresh 80% EtOH. Remove all EtOH, remove plate from magnet, and let beads dry at room temperature. DO NOT overdry beads.
(Optional) This step can be automated using TECAN Freedom Evo 100 with script - BP_1Plate_150uL_EtOH_Wash.
69. Add 20µL EB buffer and resuspend beads by pipet. Vortex and incubate for 5 minutes at room temperature, then quick spin for 10s at 2,000xg. Place back on magnet and let stand until solution is clear of beads (~5 minutes). Combine 20µL eluent from all wells in each column of the 96-well plate (8 wells per column, 12 columns) into 12 1.5µL Eppendorf tubes.
70. Add 128µL (0.8x) SPRI Beads to each 1.5µL Eppendorf tube. Pipette to mix and incubate for 5 minutes at room temperature.
71. Place 1.5µL tubes on DynaMag™-2 Magnet, let stand until solution in tubes in clear of beads (~5 minutes). Remove supernatant and wash beads 2x with 200uL fresh 80% EtOH. Remove all EtOH, remove tubes from magnet, and let beads dry at room temperature. DO NOT overdry beads.
72. Add 40µL EB and resuspend beads by pipet. Incubate for 5 minutes at room temperature. Place tubes back on magnet and let stand until solution is clear of beads (~5 minutes). Remove 40uL supernatant to 12 clean 1.5µL Eppendorf tubes.
73. Measure concentration of each 1.5µL Eppendorf tube with Qubit dsDNA BR Assay Kit. Normalize library concentrations and pool for sequencing.