TITLE: GoT-ChA: Genotyping of Targeted loci with single-cell Chromatin Accessibility
AUTHORS: Franco Izzo, Robert M Myers, Saravanan Ganesan, Levan Mekerishvili, Sanjay Kottapalli, Tamara Prieto, Elliot O Eton, Theo Botella, Ivan Raimondi, Catherine Potenski, Dan A Landau

[DESCRIPTION]
Somatic mutations are crucial for cancer initiation and evolution, and have been identified across a number of healthy tissues in the human body. These mutations can disrupt normal cellular functions, leading to aberrant clonal expansions via acquired fitness advantages or skewed differentiation topologies. GoT-ChA and similar methods (e.g. GoT, TARGET-seq) aim to pair targeted genotyping with single-cell sequencing approaches in order to understand the impact of somatic mutations directly in human patient samples, in both malignant and non-malignant contexts.
GoT-ChA is unique in that it is a high-throughput single-cell method that pairs targeted genotyping with chromatin accessibility measurements based on the broadly utilized scATAC-seq platform from 10x Genomics. Previous single-cell genotyping approaches were largely based on scRNA-seq protocols, utilizing expressed and captured mRNA transcripts as sources for genotyping information. This results in a limiting dependence on gene expression and mutation location (due to 3' end bias of most scRNA-seq methods), precluding the usage of such technologies on lowly expressed or inconveniently located loci of interest. GoT-ChA surmounts these limitations via direct utilization of genomic DNA for genotyping, whilst simultaneously assaying chromatin accessibility.

[BEFORE_START]
Designing a GoT-ChA experiment.
 

To utilize GoT-ChA, three primers need to be designed that flank the genomic region of interest. GoT-ChA_R1 (containing the partial Nextera Read1 sequence in its handle) and GoT-ChA_Rev flank the loci of interest, ideally forming an amplicon between 200-500 bps in size. GoT-ChA_nested is utilized in the hemi-nested PCR during GoT-ChA library construction, and crucially needs to bind within 50bp (inclusive of binding site) of the mutation to be covered with standard scATAC-seq sequencing parameters.

[GUIDELINES]
How does GoT-ChA work?

 
In order to capture genotypes within droplet-based scATAC-seq, two GoT-ChA primers are added into the cell barcoding PCR reaction that are designed to flank the locus of interest. One primer contains the partial Nextera Read 1N sequence in its handle, which allows for GoT-ChA amplicons to interact with the 10x Genomics gel bead oligonucleotide and obtain a unique cell barcode, just as tagmented chromatin fragments do. Further, the second GoT-ChA primer allows for exponential amplification of GoT-ChA amplicons while tagmented chromatin fragments are only linearly amplified.

 

After the single-cell emulsion is broken, a small portion of the sample is taken for GoT-ChA library construction, comprised of a hemi-nested PCR, biotin-streptavidin pull-down, and an on-bead sample indexing PCR. The final GoT-ChA library can be pooled with scATAC-seq libraries and sequenced together using standard ATAC sequencing parameters.

[STEPS]
SECTION: Buffer Preparations
1. Pre-chill swing-bucket centrifuge to 4˚C
SECTION: GoT-ChA on Permeabilized Whole Cells
6. Obtain all single cell suspensions (filter if needed) and measure viability and density (if
viability is <90% proceed with live cell enrichment and/or use best judgement
depending on sample source / importance / rarity).
SECTION: Buffer Preparations
2. Pre-heat block to 65˚C for later use dissolving digitonin

for GoT-ChA on Isolated Nuclei 
SECTION: Buffer Preparations
4. Wash buffer
 

SECTION: Buffer Preparations
3. Lysis Buffer
 



SECTION: Buffer Preparations
5. PBS + 0.04% BSA

 


SECTION: GoT-ChA on Permeabilized Whole Cells
19. Filter through 40 μm strainers
SECTION: GoT-ChA on Permeabilized Whole Cells
18. Remove supernatant, resuspend in 150 μl 1x nuclei buffer (10x Genomics)
SECTION: GoT-ChA on Permeabilized Whole Cells
17. Spin 5 minutes 500g at 4 ̊C.
SECTION: GoT-ChA on Permeabilized Whole Cells
16. Add 1 ml chilled wash buffer to the lysed cells, mix by pipetting
SECTION: GoT-ChA on Permeabilized Whole Cells
15. Incubate on ice for 3min (primary cells), 5min (cell lines)
SECTION: GoT-ChA on Permeabilized Whole Cells
14. Resuspended cell pellet in 100 μl chilled lysis buffer, mix by pipetting.
SECTION: GoT-ChA on Permeabilized Whole Cells
13. Spin 5 minutes 400g at 4 ̊C, discard supernatant.
SECTION: GoT-ChA on Permeabilized Whole Cells
12. Discard supernatant and repeat wash with 1ml 1x ice-cold PBS
SECTION: GoT-ChA on Permeabilized Whole Cells
11. Spin 5 minutes 400g at 4 ̊C.
SECTION: GoT-ChA on Permeabilized Whole Cells
10. Wash with 1x ice-cold PBS by filling up the tube, invert 5 times
SECTION: GoT-ChA on Permeabilized Whole Cells
9. Quench by adding glycine to 0.125M f.c.
SECTION: GoT-ChA on Permeabilized Whole Cells
8. Add 30 μl 16% formaldehyde (1% f.c) and incubate 10min at room temperature, swirl,
invert occasionally
SECTION: GoT-ChA on Permeabilized Whole Cells
7. Resuspend cells in 450 μl PBS.

if low cell number, scale down accordingly.
SECTION: GoT-ChA on Isolated Nuclei
23. Obtain all single cell suspensions (filter if needed) and measure viability and density (if
viability is <90% proceed with live cell enrichment and/or use best judgement
depending on sample source / importance / rarity).
SECTION: GoT-ChA on Isolated Nuclei
35. Carry with Transposition for sc-experiment using 10x kit and follow the protocol. (remember to spike in 1µL of combined Forward and Reverse (each at 22.5µM conc) to the emulsion Master Mix)

corresponding to step 2.1 from 10X genomics ATAC user guide
SECTION: GoT-ChA on Isolated Nuclei
34. Calculate amount of µL for target number of recovered cells

(max volume for loading is 5 µL for 10x transposition reaction) 
SECTION: GoT-ChA on Isolated Nuclei
33. Use 2 µL nuclei suspension mixed with 6 µL Diluted Nuclei Buffer and 2 µL trypan blue to determine nuclei concentration
SECTION: GoT-ChA on Isolated Nuclei
32. Resuspend the pellet in X µL of chilled Diluted Nuclei Buffer for a final volume close to 7-8 µL
SECTION: GoT-ChA on Isolated Nuclei
31. Remove supernatant in two steps to not touch pellet
SECTION: GoT-ChA on Isolated Nuclei
30. Spin 5 minutes at 700 x g at 4 ˚C
SECTION: GoT-ChA on Isolated Nuclei
29. Add 1 ml chilled wash buffer to the lysed cells and mix via pipetting
SECTION: GoT-ChA on Isolated Nuclei
28. Incubate on ice for 3 minutes (primary cells) or 5 minutes (cell lines)
SECTION: GoT-ChA on Isolated Nuclei
27. Resuspend cell pellet in 100 µL chilled lysis buffer and mix via pipetting
SECTION: GoT-ChA on Isolated Nuclei
25. Remove supernatant and resuspend in 100 µL room temperature PBS + BSA

if low number of cells, scale done volume
SECTION: Emulsion generation
36. As per 10x protocol. (remember to spike in Gotcha primers (22.5uM - 1ul each) per reaction during emulsion generation step) 


 


 
SECTION: SPRI Clean up
37. Continue with SPRI clean up and elute DNA with 44.5ul of EB buffer. (40ul for ATAC and 4ul for GOTCHA)

Step 3.2 from the 10X Genomics ATAC User guide
SECTION: SPRI Clean up
38. Proceed to ATAC-library process as per 10x protocol with 40ul of eluted DNA.
SECTION: Nested PCR
39. Perform 10-20 cycles of PCR with the following conditions (with nested primer)
SECTION: Nested PCR
41. GoTChA Nested PCR thermocycler program
 

SECTION: Nested PCR
40. GoTChA Nested PCR

 

SECTION: Nested PCR
42. SPRI clean up with 1.2X. Elute in 40 µL.
SECTION: Biotin- Pull Down
43. Allocate 15 µL of streptavidin M-280 beads per sample (+10%)
SECTION: Biotin- Pull Down
50. Resuspend beads in 20 µL
SECTION: Biotin- Pull Down
49. Wash one time with 100ul of 10 mM Tris HCl (pH 8.0)
SECTION: Biotin- Pull Down
48. Wash two times with 100ul of 1x SSPE buffer
SECTION: Biotin- Pull Down
47. Incubate at room temperature for 15 minutes
SECTION: Biotin- Pull Down
46. Add 10 µL of beads to 40 µL of SPRI'd sample
SECTION: Biotin- Pull Down
45. Resuspend in 5X SSPE buffer for 10 µL per sample (+10%)
SECTION: Biotin- Pull Down
44. Wash beads three times with 100ul of 1x SSPE buffer
SECTION: On-bead Sample Indexing PCR
51. Perform 6-10 cycles of PCR with the following conditions:
SECTION: On-bead Sample Indexing PCR
52. GoTChA Sample Index PCR
 

SECTION: On-bead Sample Indexing PCR
53. GoTChA Sample Index thermocycler program

 


SECTION: On-bead Sample Indexing PCR
54. Run an e-gel to assess correct product formation. Use 5 µL (10%) of total sample
SECTION: On-bead Sample Indexing PCR
55. Purify libraries using AMPureXP beads at a 1.2X ratio
SECTION: On-bead Sample Indexing PCR
56. Elute in 20 µL of EB or Water
SECTION: Assessing library quality
57. Run a BioA

Expected results: 

 







SECTION: Assessing library quality
59. Dilute samples for sequencing. GoT-ChA libraries can be sequenced together with ATAC libraries assuming i7 indexes do not clash. Sequencing parameters are as follows Read 1N - 50 cycles, i7 index -8 cycles, i5 Index - 16 cycles. Read 2N - 50 cycles.
SECTION: Assessing library quality
58. Measure samples via Qubit
SECTION: GoT-ChA on Permeabilized Whole Cells
22. Carry with Transposition for sc-experiment using 10x kit and follow the protocol. (remember to spike in 1µL of combined Forward and Reverse (each at 22.5µM conc) to the emulsion Master Mix)

corresponding to step 2.1 from 10X genomics ATAC user guide
SECTION: GoT-ChA on Isolated Nuclei
24. Spin down at 300 x g for 5 minutes at 4˚C in PCR tubes if the number of cells are very low.
SECTION: GoT-ChA on Permeabilized Whole Cells
20. Use 2 µL nuclei suspension mixed with 6 µL Diluted Nuclei Buffer and 2 µL trypan blue to determine nuclei concentration 
SECTION: GoT-ChA on Permeabilized Whole Cells
21. Calculate amount of µL for target number of recovered cells

(max volume for loading is 5 µL for 10x transposition reaction) 
SECTION: GoT-ChA on Isolated Nuclei
26. Spin down at 300 x g for 5 minutes at 4˚C