TITLE: SAMOSA-Tag - combining in-situ adenine methyltransferase footprinting with transposase-mediated single-molecule sequencing
AUTHORS: Scott Nanda, Ke   Wu, Siva Kasinathan, vijay.ramani

[DESCRIPTION]
Here we describe a protocol for SAMOSA-Tag: Tagmentation-assisted Single-molecule Adenine Methylated Oligonucleosome Sequencing Assay - a transposase-mediated strategy that combines our method SMRT-Tag (https://www.protocols.io/view/FB06B8000CA311ED84710A58A9FEAC02) for producing low-input PacBio sequencing libraries with in situ footprinting to enable multimodal profiling of single chromatin fibers. SAMOSA-Tag facilitates the joint analysis of genetic and epigenetic variations (i.e. somatic variation, nucleosome repeat length, CTCF occupancy, CpG methylation). 

In brief, nuclei are methylated in situ using the non-specific EcoGII m6dAase, tagmented using hairpin-loaded Tn5-adaptors, gap-repaired following DNA purification, and then sequenced on the PacBio Sequel II platform. All steps from live cells to sequencing-ready libraries can be performed in one to two days. We have demonstrated the utility of SAMOSA-Tag by profiling chromatin accessibility, CpG methylation, and CTCF occupancy on low cell numbers in mammalian cell lines. 



  


[GUIDELINES]
Sensitive multimodal profiling of native DNA by transposase-mediated single-molecule sequencing

Arjun S Nanda1,2*, Ke Wu1*, Sivakanthan Kasinathan3,4*#, MeganSOstrowski1, Andrew S Clugston5,6, Ansuman T Satpathy4,7,8, E Alejandro Sweet-Cordero5,6, Hani Goodarzi2,6,8,9, Vijay Ramani1,2,6,9#

1Gladstone Institute for Data Science & Biotechnology, Gladstone Institutes, San Francisco, CA 94158
2Department of Biochemistry & Biophysics, UCSF, San Francisco, CA 94143
3Division of Allergy, Immunology, and Rheumatology, Department of Pediatrics, Stanford University, Stanford, CA 94305
4Gladstone-UCSFInstituteforGenomic Immunology, Gladstone Institutes, San Francisco, CA 94143
5Department of Pediatrics, UCSF, San Francisco, CA 94143
6Helen-Diller Cancer Center, San Francisco, CA 94158
7Department of Pathology, Stanford University, Stanford, CA 94305
8Parker Institute for Cancer Immunotherapy, San Francisco, CA 94129
9Bakar Computational Health Sciences Institute, San Francisco,CA 94143
*These authors contributed equally to this work.
#correspondence to skas@stanford.edu or vijay.ramani@gladstone.ucsf.edu


[STEPS]
SECTION: Reagent Setup
1. Nuclear Lysis Buffer (prepared on ice): 
 

Buffer M (prepared on ice):
 

Nuclei Storage Buffer (prepared on ice):
 

5x TD Premix (prepared at RT):
 


SMRT-Tn5 adaptor assembly:
we recommend following the "Annealing SMRT-Tag adaptors" and "Assembling SMRT-Tn5 transposomes (Tn5 loaded with SMRT-Tag adaptors)" procedures in SMRT-Tag protocol:  https://www.protocols.io/view/FB06B8000CA311ED84710A58A9FEAC02

SECTION: Nuclei isolation from fresh cells
2. Harvest fresh cell culture(s) in a conical centrifuge tube at room temperature and count cells. This protocol can accommodate up to a total of 2 million mammalian cells (e.g. human K562, murine embryonic stem cells) that can be divided into 10-30k nuclei per sample for up to 16 samples to be sequenced.
SECTION: Nuclei isolation from fresh cells
3. Centrifuge a 1 to 2 million cell suspension at 300xg at 4ºC for 5 min, and aspirate the supernatant. 

[NOTE] For robust cell lines including cancer cell lines, 10 minutes of centrifugation can increase recovery.
SECTION: Nuclei isolation from fresh cells
4. Resuspend the cell pellet in 1mL ice-cold Nuclear Lysis Buffer. Pipet up and down gently 10x using wide-bore tips. 
(Use approximately 1mL Nuclear Lysis Buffer for up to 10 million cells.)
SECTION: Nuclei isolation from fresh cells
5. Incubate on ice for exactly 5 min.
SECTION: Nuclei isolation from fresh cells
6. Centrifuge at 600xg at 4ºC for 5 min, and aspirate the supernatant. 

[NOTE] For robust cell lines including cancer cell lines, 10 minutes of centrifugation can increase recovery.
SECTION: Nuclei isolation from fresh cells
7. Wash nuclei with 1mL Buffer M.

[cell count 0] Determine # of nuclei from a 10 µL aliquot (1:1 dilution) via hemocytomer / cell counter (10 µL nuclei suspension + 10 µL trypan blue)
SECTION: Nuclei isolation from fresh cells
8. Centrifuge again at 600xg at 4ºC for 5 min, and aspirate the supernatant. 

[NOTE] For robust cell lines including cancer cell lines, 10 minutes of centrifugation can increase recovery.
SECTION: Nuclei isolation from fresh cells
9. Proceed immediately to nuclei freezing or EcoGII methylation. 
SECTION: (Optional) Nuclei freezing
10. Isolated nuclei (from step 8) may be slowly frozen by resuspending in nuclei freezing media containing 9 volumes of Nuclei Storage Buffer and 1 volume of DMSO, and further divided into 1 mL per cryogenic vial (~1-2 million nuclei), then stored at -80°C. We have found a cooling rate close to -1°C/min is sufficient for preserving nuclei integrity. 
SECTION: (Optional) Thawing previously frozen nuclei
11. If frozen nuclei are used as input, thaw ~1-2 million nuclei in a water bath at 37ºC for 1-2 min until most of the solution is a slurry. Expect 30-50% recovery after each wash step. This protocol is designed for up to 1-2 million frozen nuclei that can be further divided into 10-30k nuclei per sample for up to 16 samples to be sequenced but can be adapted for lower numbers of nuclei per replicate, or fewer replicates, as needed.
 
[CRITICAL] Nuclei are thawed in 10% DMSO and need to be washed rapidly in order to prevent lysis. 
SECTION: (Optional) Thawing previously frozen nuclei
12. Use a P1000 with wide-bore pipette tips to gently transfer nuclei solution to a 1.5mL LoBind tube. 
SECTION: (Optional) Thawing previously frozen nuclei
13. Centrifuge at 600xg at 4ºC for 5 min, and aspirate the supernatant. Leave a tiny volume around the pellet is fine.

[NOTE] For robust cell lines including cancer cell lines, 10 minutes of centrifugation can increase recovery.

SECTION: (Optional) Thawing previously frozen nuclei
14. Resuspend pelleted nuclei in 1mL Buffer M by gentle pipetting using a P1000 with wide-bore tips. 

[cell count 1] Determine # of nuclei from a 10 µL aliquot (1:1 dilution) via hemocytomer / cell counter (10 µL nuclei suspension + 10 µL trypan blue)
SECTION: (Optional) Thawing previously frozen nuclei
15. Centrifuge again at 600xg at 4ºC for 5 min, and aspirate the supernatant. 

[NOTE] For robust cell lines including cancer cell lines, 10 minutes of centrifugation can increase recovery.
SECTION: (Optional) Thawing previously frozen nuclei
16. Proceed immediately to EcoGII methylation. 
SECTION: in-situ SAMOSA (or EcoGII Methylation)
17. Resuspend the nuclei (either freshly isolated nuclei from step 8 or previoiusly-frozen nuclei from step 15) in 400 µL  buffer containing Buffer M + 1mM SAM (e.g. 12.5 µL of 32mM SAM + 387.5 µL Buffer M).  

[cell count 2] Determine # of nuclei from a 10 µL aliquot (1:1 dilution) via hemocytomer / cell counter (10 µL nuclei suspension + 10 µL trypan blue)
SECTION: in-situ SAMOSA (or EcoGII Methylation)
18. Split solution into 2 x 200 µL reactions, one for methylated (+M) and the other for unmethylated  (-M) nuclei.

[NOTE] If processing a sample where unmethylated data has been previously obtained, or samples are precious, the unmethylated condition can be skipped.

[PAUSE]  Determine if you have enough nuclei for downstream processing, assuming 50% loss after EcoGII methylation. (i.e 8 samples x 2 methylation states x 30k per rxn = ~ 500K nuclei required)

SECTION: in-situ SAMOSA (or EcoGII Methylation)
19. Add 10 µL of EcoGII (25U/µL) to +M rxn. For -M rxn, do not add EcoGII. Mix well by slowly pipetting with wide-bore tips or gently flicking to mix.


SECTION: in-situ SAMOSA (or EcoGII Methylation)
20. Incubate both reactions at 37ºC for 30 min in a thermomixer with 300rpm shaking every 2 min. At the 15 min time point, supplement with 1 µL of 32mM SAM stock in both reactions.
SECTION: in-situ SAMOSA (or EcoGII Methylation)
21. Terminate the reactions by placing them back on ice.
SECTION: Tagmentation of SAMOSA-treated nuclei
22. Prepare Omni-ATAC Reagent Mix on ice. Prepare a little extra (5 samples worth) for sample dilution.

 

SECTION: Tagmentation of SAMOSA-treated nuclei
23. Centrifuge 2 x 200 µL reactions after EcoGII methylation at 600xg at RT for 10 min, and carefully pipette to remove the supernatant. Leave a tiny volume around the pellet is fine.

[CRITICAL] Nuclei may be smeared out across the LoBind tube wall instead of nicely pelleted at the bottom.
SECTION: Tagmentation of SAMOSA-treated nuclei
24. Resuspend nuclei gently in 250 µL Omni-ATAC Reagent Mix per reaction. 
SECTION: Tagmentation of SAMOSA-treated nuclei
25. Using a P1000 with a wide-bore tip, set the pipette to 300 µL and aspirate up the entire volume per reaction. Insert the tip into a FlowMi cell strainer, and then slowly push the reaction through the strainer into a new LoBind tube. There will be bubbles ejecting upward, so make sure filtered tips are used. 

[cell count 3] Determine # of nuclei post filtration from a 10 µL aliquot (1:1 dilution) via hemocytomer / cell counter (10 µL nuclei suspension + 10 µL trypan blue) for both +M and -M reactions

[CRITICAL] QC check - if nuclei still look clumped together, make a note - your count is likely NOT accurate, and may require adjusting the Tn5 amount or nuclei input per sample. Nuclei clumping tends to underestimate the actual count number. 
SECTION: Tagmentation of SAMOSA-treated nuclei
26. Split each of the +M and -M reactions into multiple tubes as needed such that ~ 10K - 30K nuclei are in each tube.

[CRITICAL] The majority of samples to be sequenced should be +M which are used to profile chromatin accessibility.
SECTION: Tagmentation of SAMOSA-treated nuclei
27. To generate an average of 3kb libraries, add 2 µL of loaded SMRT-Tn5 adaptor stock (~ 9.4µM monomer) per sample and fill the total volume up to 50 µL with Omni-ATAC reagent mix.

[OPTIONAL] QC check - Prepare 1 untagged negative control for each of +M and -M reactions to compare against tagged samples. A total of two negative controls: +M -tag and -M -tag. Replace SMRT-Tn5 adaptor volume with Omni-ATAC reagent mix. 
SECTION: Tagmentation of SAMOSA-treated nuclei
28. Incubate all tagmentation reactions at 55ºC for 1 hr and hold at 4ºC. 
SECTION: Tagmentation termination
29. Treat the tagmented nuclei with 10 µL of RNase A (10ug/µL) and incubate at 37ºC for 15 min on a thermomixer with 300rpm mixing every 2 min.

[CRITICAL] RNAse pre-treatment is critical to ensure that only DNA is extracted from the reaction.
SECTION: Tagmentation termination
30. Prepare the Termination Lysis Reagent Mix about 10 min before the tagmentation is complete.

 

[CRITICAL] Equilibriate Proteinase K at RT before adding to the mixture. Do NOT keep it on ice, or the SDS mixture will precipitate with EDTA. Vortex EDTA stock solution prior to use. If the mixture appears cloudy, warm it up at RT until it appears homogenous again. 
SECTION: Tagmentation termination
31. After RNase treatment, add 7.5 µL of termination lysis reagent mix to each sample and mix well.
SECTION: Tagmentation termination
32. Incubate at 60ºC on a thermomixer with 1000rpm continuous shaking for at least 1 hr, up to 2 hr for improved lysis.
SECTION: gDNA purification with SPRI cleanup
33. Remove samples from thermomixer and incubate on ice for 1 min to cool down samples to RT.
SECTION: gDNA purification with SPRI cleanup
34. Add 2x volume of resuspended, room-temperature SPRI beads. (i.e. For a 50 µL tagmentation reaction, sample volume should be 50 µL tagmentation reaction + 10 µL RNase A + 7.5 µL termination lysis MM ~ 67.5 µL, so 2X ~ 135 µL). Mix well using a P200 with wide-bore tips.
SECTION: gDNA purification with SPRI cleanup
35. Incubate the bead-mixed samples in a thermomixer at 23ºC for 30 min with interval mixing @350 rpm (1 min on, 3 min off) to keep the beads resuspended.
SECTION: gDNA purification with SPRI cleanup
36. Spin down quickly. Place on magnet and allow to clear before carefully withdrawing supernatant. 
SECTION: gDNA purification with SPRI cleanup
37. Wash beads twice with 80% ethanol for 30 sec. Add ethanol to the wall opposite of beads. Withdraw supernatant and after a quick spin, remove the remaining liquid with a P20 pipette. Beads take ~ 30 s to 1 min to dry. Do not over-dry the beads - dried beads appear to be fragmented with cracks in light brown. 
SECTION: gDNA purification with SPRI cleanup
38. Remove from the magnet stand, and gently resuspend the bead pellet in 20 µL 1x EB (or 10 mM Tris-HCl, pH 8.5) to elute the extracted DNA. Once beads are resuspended, mix well. 
SECTION: gDNA purification with SPRI cleanup
39. Incubate the samples in a Thermomixer at 37ºC for 15 min with interval mixing @350 rpm (1 min on, 3 min off) to promote increased DNA elution. 
SECTION: gDNA purification with SPRI cleanup
40. Spin samples down quickly. Place on magnet and allow samples to clear before carefully transferring the supernatant to a new LoBind tube. 

[Pause] Purified gDNA samples can be stored at 4ºC for up to two weeks.
SECTION: gDNA purification with SPRI cleanup
41. Determine concentrations of purified DNA using Qubit 1x High Sensitivity DNA Assay. Expect at least 160ng of DNA recovered from 10-30k nuclei. 
SECTION: gDNA purification with SPRI cleanup
43. [Optional] QC check - Visualize the SMRT-Tn5 transposome efficiency by running 2 untagged controls against tagged samples on a 0.5% agarose gel coupled with the NEB 1kb-Extend ladder. Expect to see the majority of bands clustered towards high molecular weight >48.5kb for untagged samples, whereas a long smear should be observed for tagged samples. Untagged samples will not be sequenced as they do not contain SMRT-Tag adaptors.  
SECTION: Gap Repair
44. Prepare the Gap-Repair Reagent Mix.

 


SECTION: Gap Repair
45. Incubate the gap-repair reactions at 37ºC for 1hr and hold at 4ºC.
SECTION: Gap Repair
46. Add 2x volume of resuspended, room-temperature SPRI beads to clean up the reactions. Mix well using a P200 with wide-bore tips.

SECTION: Exonuclease Digestion
54. Prepare the Exo-Digest Reagent Mix.

 

SECTION: Gap Repair
53. [Optional] QC check - take 1 µL aliquot and determine concentrations of gap-repaired samples using Qubit 1x High Sensitivity DNA Assay. Expect a 70% or above recovery from tagmented sample input in step 44. 
SECTION: Exonuclease Digestion
55. Incubate the exo-digested reactions at 37ºC for 1hr and hold at 4ºC.
SECTION: Library QC and Sequencing
64. Pool barcoded libraries equimolarly and sequence on PacBio Sequel II 8M SMRTcells. We recommend using Sequel II Binding Kit 2.1 for the best loading result, as well as a 30h movie time, 2h pre-extension, and 4h immobilization time. 

SECTION: gDNA purification with SPRI cleanup
42. [OPTIONAL] Determine the fragment size distribution using Agilent 2100 Bioanalyzer High Sensitivity DNA Assay.

 
 

[OPTIONAL] If BioAnalyzer trace shows a trailing tail <1kb (e.g. reference fig. 1), we recommend doing an additional 0.6x Ampure PB cleanup to enrich for fragments > 500bp.

SECTION: Library QC and Sequencing
63. Determine library concentration using Qubit 1x High Sensitivity DNA Assay. 

Determine the size distribution using Agilent 2100 Bioanalyzer High Sensitivity DNA Assay. 

[CRITICAL] Convert the BioA trace from its default unit (fluorescent unit) to "molarity per length" using the R package "bioanalyzeR". This step is critical for the evaluation of accurate library sizing required for the optimal loading on PacBio Sequel II. 
 


 


SECTION: Gap Repair
47. Incubate the bead-mixed samples in a thermomixer at 23ºC for 15 min with interval mixing @350 rpm (1 min on, 3 min off) to keep the beads resuspended.
SECTION: Gap Repair
48. Spin down quickly. Place on magnet and allow to clear before carefully withdrawing supernatant. 
SECTION: Gap Repair
49. Wash beads twice with 80% ethanol for 30 sec. Add ethanol to the wall opposite of beads. Withdraw supernatant and after a quick spin, remove the remaining liquid with a P20 pipette. Beads take ~ 30 s to 1 min to dry. Do not over-dry the beads - dried beads appear to be fragmented with cracks in light brown. 
SECTION: Gap Repair
50. Remove from the magnet stand, and gently resuspend the bead pellet in 12 µL 1x EB (or 10 mM Tris-HCl, pH 8.5) to elute the extracted DNA. Once beads are resuspended, mix well. 
SECTION: Gap Repair
51. Incubate the samples in a Thermomixer at 37ºC for 10 min with interval mixing @350 rpm (1 min on, 3 min off) to promote increased DNA elution. 
SECTION: Gap Repair
52. Spin samples down quickly. Place on magnet and allow samples to clear before carefully transferring the supernatant to a new LoBind tube. 

SECTION: Exonuclease Digestion
56. Add 2x volume of resuspended, room-temperature SPRI beads to clean up the reactions. Mix well using a P200 with wide-bore tips.

SECTION: Exonuclease Digestion
57. Incubate the bead-mixed samples in a thermomixer at 23ºC for 15 min with interval mixing @350 rpm (1 min on, 3 min off) to keep the beads resuspended.
SECTION: Exonuclease Digestion
58. Spin down quickly. Place on magnet and allow to clear before carefully withdrawing supernatant. 
SECTION: Exonuclease Digestion
59. Wash beads twice with 80% ethanol for 30 sec. Add ethanol to the wall opposite of beads. Withdraw supernatant and after a quick spin, remove the remaining liquid with a P20 pipette. Beads take ~ 30 s to 1 min to dry. Do not over-dry the beads - dried beads appear to be fragmented with cracks in light brown. 
SECTION: Exonuclease Digestion
60. Remove from the magnet stand, and gently resuspend the bead pellet in 12 µL 1x EB (or 10 mM Tris-HCl, pH 8.5) to elute the extracted DNA. Once beads are resuspended, mix well. 
SECTION: Exonuclease Digestion
61. Incubate the samples in a Thermomixer at 37ºC for 10 min with interval mixing @350 rpm (1 min on, 3 min off) to promote increased DNA elution. 
SECTION: Exonuclease Digestion
62. Spin samples down quickly. Place on magnet and allow samples to clear before carefully transferring the supernatant to a new LoBind tube.