TITLE: SNARE-seq2 with Nuclei Hashing 
AUTHORS: Nongluk Plongthongkum, Dinh H Diep, Song Chen, Blue Lake, Kun Zhang

[DESCRIPTION]
To study the heterogeneity of complex tissues by joint profiling of gene expression and its regulation, we require an accurate and high-throughput method. Here we described improved high-throughput combinatorial indexing-based single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-Seq2) co-assay. This protocol involves fixing and permeabilizing the nucleus followed by tagmentation, chromatin barcode ligation, reverse transcription, pooling and splitting for the next rounds of cell barcode ligation into cDNA and accessible chromatin (AC) on the same nucleus. The captured cDNA and AC are co-amplified before splitting and enrichment into single-nucleus RNA and single-nucleus AC sequencing libraries. The protocol can also be applied to both nuclei and whole cells to capture mRNA in the cytoplasm. This improvement allows us to generate hundreds of thousands of data set of each assay and can be scaled up to half a million cells from a single experiment. The entire procedure can be complete in 3.5 d for generating joint single-nucleus RNA and single-nucleus ATAC sequencing libraries.

[STEPS]
SECTION: Reagent setup
1. 40% (wt/vol) PEG 6000. Weigh 16.0 g of PEG 6000 in 50 mL tube. Add nuclease-free water and bring the total volume to 40 mL. Rotate the tube at room temperature until PEG 6000 completely dissolved. Spin down the tube at 200 g for 2 min, at room temperature to remove the tiny bubble. CRITICAL: 40% (wt/vol) of PEG 6000 is very viscous and difficult to filter through a 0.22 µm filter. We suggest preparing 40% PEG freshly before making GLR buffer. When PEG is diluted in 4x GLR buffer, it is easier to filter.
SECTION: Reagent setup
2. 4x GLR buffer. To prepare 40 mL of 4x GLR buffer, Add 2.64 mL of nuclease-free water, 10.56 mL of 1 M Tris-HCl, pH 8.0, 0.8 mL of 1 M MgCl2 and 4 mL of 2 M KCl into 50 mL tube. Gently mix well by vortexing. Add 22 mL of 40% (wt/vol) PEG 6000 and gently mix well by vortexing. Filter through 0.22 µm into a new 50 mL tube and briefly spin the tube at room temperature for 30 s. Aliquot 1.8 mL into 2 mL tubes to minimize contamination from each use and store at 4 ºC.
SECTION: Reagent setup
3. 10% (vol/vol) Triton X-100. Slowly aspirate 2.0 mL of Triton X-100 liquid with low retention pipette tip and slowly dispend into 18.0 mL nuclease-free water in 50 mL tube. Dissolve Triton X-100 by slowly rotate the tube until the solution is clear. Filter 10% Triton X-100 solution through 0.22 µm syringe filter into a new 50 mL tube and store at room temperature. CRITICAL: If it’s difficult to pipette Triton X-100 accurately as it’s a viscous liquid, we may warm it at 37 ºC before pipetting
SECTION: Reagent setup
4. 10% (vol/vol) Tween 20. Tween 20 is very viscous liquid and difficult to pipette accurately. We convert the volume in cm3 into grams using the density of tween 20 at 25 ºC is 1.1 cm3. To prepare 20 mL of 10% (vol/vol) Tween 20, weigh 2.2 g of Tween 20 in 50 mL tube. Add 18.0 mL of nuclease-free water and invert or rotate the tube slowly at room temperature until Tween 20 is completely dissolved in water. Filter 10% Tween 20 solution through 0.22 µm syringe filter into a new 50 mL tube and store at room temperature.
SECTION: Reagent setup
5. 25 mM dNTP mix. Mix 250 µL each of 100 mM dATP, dCTP, dGTP and dTTP in 1.5 mL tube. Mix well by vortexing and briefly spin the tube at room temperature for 5 s. Aliquot 250 µL into each of 1.5 mL tube and store at -20 ºC for a couple of years
SECTION: Reagent setup
6. 2x Lysis buffer. To prepare 25 mL of 2x Lysis buffer, add 6.5 mL of nuclease-free water into 50 mL tube. Add 0.5 mL of 1 M Tris-HCl, pH 8.0, 2 mL of 5 M NaCl, 5 mL of 0.5 M EDTA and 11 mL of 10% (wt/vol) SDS. Gently mix and aliquot 1.8 mL into 2 mL tubes and store at room temperature.
SECTION: Reagent setup
7. 1x B&W-T buffer. To prepare 40 mL of 1x B&W-T, add 31.56 mL of nuclease-free water into 50 mL tube. Add 200 µL of 1 M Tris-HCl, pH 8.0, 8 mL of 5 M NaCl, 40 µL of 0.5 M EDTA and 200 µL of 10% (vol/vol) Tween 20. Gently mix by vortexing and store at room temperature.
SECTION: Reagent setup
8. 2x B&W buffer. To prepare 25 mL of 1x B&W, add 14.7 mL of nuclease-free water into 50 mL tube. Add 250 µL of 1 M Tris-HCl, pH 8.0, 10 mL of 5 M NaCl, and 50 µL of 0.5 M EDTA. Mix well by vortexing and store at room temperature.
SECTION: Reagent setup
9. 0.1 M PMSF. Weigh 34.8 mg of PMSF and transfer into 2 mL microtube. Add 100% isopropanol to 2 mL and vortex vigorously to dissolve PMSF. Quick spin the tube down and transfer all solution into 3 mL syringe. Filter through a 0.22 µm syringe filter to a new 2 mL tube. Aliquot 50 µL per 0.2 mL PCR tube and store at -20 ºC for up to 4 months. CRITICAL: To maintain the activity of PMSF in solution, store PMSF in single-use aliquots.
SECTION: Reagent setup
10. Transposon preparation. Resuspend Nextera adapter 1, 5P-Nextera adapter 2 and mosaic end (ME) oligos with nuclease-free water to 100 µM. Mix 500 µL of 100 µM Nextera adapter 1 and 500 µL of 100 µM ME in 1.5 mL DNA LoBind microtube, aliquot 30 µL of non-annealed transposons into each of 0.2 mL PCR tube and store at -20 ºC for up to 1-2 years. Prepare 5P-Nextera adapter 2 and ME the same way as Nextera adapter 1. All sequences of oligos can be found in the attached tables.
SECTION: Round 1 DNA barcoding plates generation
12. Accessible chromatin (AC) Round 1 barcoded oligos preparation (Plate A). Prepare 50 µL of 25 µM accessible chromatin (AC) Round 1 barcoded oligos annealed with 23 µM accessible chromatin (AC) Round 1 linker oligos (BC_0100)
SECTION: Round 1 DNA barcoding plates generation
12.1. Resuspend AC Round 1 linker (BC_0100) with nuclease-free water to final concentration of 1 mM
SECTION: Round 1 DNA barcoding plates generation
12.2. Prepare 2.5 mL of 30.67 µM AC Round 1 linker by adding 76.68 µL of 1 mM of AC Round 1 linker to 2,423.32 µL of nuclease-free water in 5 mL tube and mix well by vortexing
SECTION: Round 1 DNA barcoding plates generation
12.3. Add 12.5 µL of 100 µM AC Round 1 barcoded oligos into 96-well plate (total 48 wells, rows A - D) with multichannel pipette
SECTION: Round 1 DNA barcoding plates generation
12.4. Transfer AC Round 1 linker oligos into 25 mL reservoir
SECTION: Round 1 DNA barcoding plates generation
12.5. Add 37.5 µL of 30.67 µM AC Round 1 linker into each well of AC Round 1 barcodes with multichannel pipette and mix well by pipetting 12 times (mixing volume 45 µL)
SECTION: Round 1 DNA barcoding plates generation
12.6. CRITICAL STEP: Seal and spin down the plate on 96-well plate swinging bucket rotor at 160 g for 1min, 4 ºC. 
SECTION: Round 1 DNA barcoding plates generation
12.7. Anneal AC Round 1 barcoded oligos and AC linker oligos on thermocycler using the following annealing program: 95 ºC for 2 min, slowly cool down to 20 ºC (0.1 ºC/s) and hold at 4 ºC. CRITICAL STEP: After annealing oligonucleotides in the plate, we recommend spinning down the plate and use a sterile needle to punch the holes on the sealing film to release the pressure in every single well. Otherwise, the liquid inside the well will be pulled up to the top of the well when the film is unsealed, and this can lead to barcode cross-contamination. PAUSE POINT: If do not want to continue to mix these annealed oligos in the next step, store that plate at -20 ºC 
SECTION: Round 1 DNA barcoding plates generation
13. RNA reverse transcription (RT) Round 1 barcoded oligos preparation (Plate B). Prepare 50 µL of 25 µM of oligo (dT)15 and 25 µM of random hexamer (N6) RT barcoded oligos (see attached tables) mix in each of 48 wells. CRITICAL STEP: 100 µM of dT and N6 reverse transcription barcoded oligos are ordered in row A-D and row E-H of oligo plate, respectively.
SECTION: Round 1 DNA barcoding plates generation
13.1. Transfer 12.5 µL of 100 µM of rows A-D (dT) in the RT barcoded oligos plate to rows A-D of a new 96-well DNA LoBind plate. CRITICAL STEP If use electronic multichannel pipette, add 1 µL of air after aspirating to avoid cross-contamination of barcoded oligos. This can be applied to other steps when we have to transfer barcoded oligos from stock plate to a new plate. Make sure that oligos are delivered to the bottom of the well.
SECTION: Round 1 DNA barcoding plates generation
13.2. Transfer 12.5 µL of 100 µM of rows E-H oligos (N6) in RT barcoded oligos plate to rows A-D (row E to row A, row F to row B, row G to row C, row H to row D) of 96-well plate that contains dT barcoded oligos above
SECTION: Round 1 DNA barcoding plates generation
13.3. Pipette 2 mL of nuclease-free water into 25 mL reservoir
SECTION: Round 1 DNA barcoding plates generation
13.4. Add 25 µL of nuclease-free water to row A-D of RNA Round 1 stock plate and mix well by pipetting 12 times (mixing volume 45 µL) 
SECTION: Round 1 DNA barcoding plates generation
13.5. Seal and spin down the plate at 160 g for 1 min, 4 ºC and leave the plate on ice or PCR cool rack
SECTION: Round 1 DNA barcoding plates generation
14. AC Round 1 barcoded oligos and RT Round 1 barcoded oligos mix. The final concentration of each oligo (dT, N6, AC) in the oligo mix is 12.5 µM.
SECTION: Round 1 DNA barcoding plates generation
14.1. Transfer 50 µL of oligos in plate B (RT Round 1 barcoded oligos) into plate A that contain 50 µL of annealed AC Round 1 barcoded oligos and linker at identical well IDs (rows A-D) and mix well by pipetting 12 times (mixing volume 90 µL)

SECTION: Round 1 DNA barcoding plates generation
14.2. Spin down the plate at 160 g for 1 min, 4 ºC and put the plate on PCR cool rack
SECTION: Round 1 DNA barcoding plates generation
14.3. Aliquot 4 µL of mixed Round 1 barcoded oligos (rows A-D) into 10-25 of 96-well plates as “working plates”
SECTION: Round 1 DNA barcoding plates generation
14.4. Spin down working plates at 160 g for 1 min, 4 ºC and store at -20 ºC for up to a couple of years. Store the left over Round 1 stock plate at -20 ºC.
SECTION: Round 2 DNA barcoding plates generation
15. Ligation Round 2 barcoded oligos. Prepare stock plate of 100 µL of 18 µM ligation Round 2 barcoded oligos annealed with 16.5 µM ligation Round 2 linker (BC_0215).
SECTION: Round 2 DNA barcoding plates generation
16. Resuspend ligation Round 2 linker (BC_0215) with nuclease-free water to final concentration of 1 mM
SECTION: Round 2 DNA barcoding plates generation
17. Prepare 9 mL of 20.12 µM ligation Round 2 linker by adding 181.08 µL of 1 mM round 2 linker to 8,818.9 µL of nuclease-free water in 15 mL tube and mix well by vortexing
SECTION: Round 2 DNA barcoding plates generation
18. Add 18 µL of 100 µM of ligation round 2 barcoded oligos into 96-well plate (rows A-H)
SECTION: Round 2 DNA barcoding plates generation
19. Transfer ligation Round 2 linker into 25 mL reservoir
SECTION: Round 2 DNA barcoding plates generation
20. Add 82 µL of 20.12 µM ligation Round 2 linker to each well of ligation Round 2 barcoded oligos with and mix well by pipetting 10 times (mixing volume 90 µL)
SECTION: Round 2 DNA barcoding plates generation
21. Seal and spin down the plate at 160 g for 1min, 4 ºC
SECTION: Round 2 DNA barcoding plates generation
22. Anneal ligation Round 2 barcoded oligos and Round 2 linker on thermocycler using the annealing program for Round 1 barcoded oligos and keep on ice
SECTION: Round 2 DNA barcoding plates generation
23. Spin down the plate at 160 g for 1min, 4 ºC and keep on ice
SECTION: Round 2 DNA barcoding plates generation
24. Aliquot 10 µL of annealed ligation Round 2 barcoded/linker oligos into 10 of 96-well plate as “working plate”
SECTION: Round 2 DNA barcoding plates generation
25. Spin down the working plate at 160 g for 1min, 4 ºC before store at -20ºC for up to a couple of year
SECTION: Round 3 DNA barcoding plates generation
26. Ligation Round 3 barcoded oligos. Prepare stock plate of 100 µL of 21 µM Round 3 barcoded oligos annealed with 19.5 µM ligation Round 3 linker (BC_0060).
SECTION: Round 3 DNA barcoding plates generation
27. Resuspend ligation Round 3 linker (BC_0060) with nuclease-free water to final concentration of 1 mM
SECTION: Round 3 DNA barcoding plates generation
28. Prepare 8.5 mL of 24.68 µM ligation Round 3 linker by adding 209.8 µL of 1 mM Round 3 linker to 8,290.2 µL of nuclease-free water in 15 mL tube and mix well by vortexing
SECTION: Round 3 DNA barcoding plates generation
29. Add 21 µL of 100 µM of ligation Round 3 barcoded oligos into 96-well plate (rows A-H)
SECTION: Round 3 DNA barcoding plates generation
30. Transfer ligation Round 3 linker into 25 mL reservoir
SECTION: Round 3 DNA barcoding plates generation
31. Add 79 µL of 24.68 µM ligation Round 3 linker to each well of ligation Round 3 barcoded oligos and mix well by pipetting 12 times (mixing volume 90 µL)
SECTION: Round 3 DNA barcoding plates generation
32. Seal and spin down the plate at 160 g for 1min, 4 ºC
SECTION: Round 3 DNA barcoding plates generation
33. Anneal ligation Round 3 barcoded oligos and Round 3 linker on thermocycler using the annealing program for Round 1 barcoded oligos and keep on ice
SECTION: Round 3 DNA barcoding plates generation
34. Spin down the plate at 160 g for 1min, 4 ºC and keep on ice
SECTION: Round 3 DNA barcoding plates generation
35. Aliquot 10 µL of annealed ligation Round 3 barcoded/linker oligos into 10 of 96-well plate as “working plate”
SECTION: Round 3 DNA barcoding plates generation
36. Spin down the working plate at 160 g for 1min, 4 ºC before store at -20ºC for up to a couple of year
SECTION: Transposase preparation
37. 
 

SECTION: Transposase preparation
38. Thaw non-annealed transposons (Nextera adapter 1/ME and 5P-Nextera adapter 2/ME) on ice. Briefly vortex and quick spin the tube. Anneal transposons on thermocycler using following program: 95 ºC 5 min, slowly cool down to 65 ºC (0.1 ºC/s), 65 ºC 5 min, slowly cool down to 4 ºC (0.1 ºC/s) and hold at 4 ºC.
SECTION: Transposase preparation
39. Load transposons 1.5x molar ratio to Tn5. The amount below is sufficient for tagmenting ~ 1.8 million nuclei/cells.
13 µL 
7.35 µL 
7.35 µL 
39 µL 
Add annealed Nextera adapter 1/ME and annealed 5P-Nextera adapter 2/ME into the bottom of 1.5 mL DNA LoBind microtube, mix well by pipetting 10 times or gently vortexing and briefly spin the tube on mini centrifuge for 3 s. Addunloaded Tn5 and mix by gently pipetting 20 times (set the volume of p200 pipette to 80 µL). Quick spin the tube and incubate at 25 ºC for 0.5 h, 350 rpm. The final concentration of loaded Tn5 is 7.35 µM (monomer Tn5 concentration).
30 min 

SECTION: Nuclei isolation and fixation
40. Isolate nuclei from tissue following tissue-specific nuclei extraction protocol (dx.doi.org/10.17504/protocols.io.ufketkw) with 0.1 U/µL of SUPERase In RNase Inhibitor and 0.2 U/µL of Enzymatics RNase Inhibitor. For cell lines, nuclei can be extracted with ATAC Lysis buffer with 0.1% NP-40 as previously described with the addition of RNase inhibitors, and increase lysis volume proportional to the number of input cells.

SECTION: Nuclei isolation and fixation
42. Prepare 1x PBS + RI (1 mL per sample) and keep on ice.
1000 µL 
2.5 µL 
1.25 µL 

SECTION: Nuclei isolation and fixation
43. Prepare 1% (wt/vol) formaldehyde in 1x PBS (1 mL per sample) and keep on ice. CRITICAL: formaldehyde solution should be in 1x PBS and methanol free.
937.5 µL 
62.5 µL 

SECTION: Nuclei isolation and fixation
44. Resuspend 1-2 million nuclei with 1 mL 1x PBS + RI and keep on ice. To each tube of 1 mL volumes of nuclei suspensions, add 15 µL of hashing oligos. Make a note of which oligo was added to which sample. Mix briefly.
SECTION: Nuclei isolation and fixation
45. Add 1 mL of 1% formaldehyde to nuclei suspension and pipette gently 8 times. Leave the tube on ice for 10 minutes.
10 min 

SECTION: Nuclei isolation and fixation
46. Pellet nuclei at 900 g for 8 min at 4C using bucket rotor centrifuge.
SECTION: Nuclei isolation and fixation
47. Prepare 1x PBS + 0.1% (wt/vol) BSA + RI ( 1 mL per sample) and leave on ice.
1000 µL 
13.4 µL 
1.5 µL 
0.75 µL 

SECTION: Nuclei isolation and fixation
48. Prepare 1x Tango Buffer + RI (1 mL per ~ 3 million nuclei/cells) and leave on ice.
100 µL 
160 µL 
5 µL 
2.5 µL 
732.5 µL 

SECTION: Nuclei isolation and fixation
49. Aspirate the supernatant, resuspend pelleted nuclei with 0.1 mL of 1x PBS + 0.1% BSA + RI to wash. Pool up to 8 samples, ~800 uL total max. Add wash buffer to 1 mL total volume.
SECTION: Nuclei isolation and fixation
50. Pellet nuclei at 900 g for 8 min at 4C using bucket rotor centrifuge.
SECTION: Nuclei isolation and fixation
51. Aspirate the supernatant and resuspend with 1x Tango Buffer + RI to have a minimum concentration of 3,400 nuclei per microliter.
SECTION: Nuclei isolation and fixation
52. Count the nuclei using cell counter and resuspend the nuclei solution with additional 1x Tango Buffer + RI to obtain 3,400 nuclei per microliter.
SECTION: Tagmentation
53. Set up tagmentation mix per reaction as follows (1 reaction per 150,000 nuclei/cells). Minimum 4 reactions for 1 sample per plate (48 wells of round 1).
3 µL 
4.8 µL 
4.95 µL 
1.5 µL 
0.75 µL 
15 µL 
SECTION: Tagmentation
54. Prepare 150,000 nuclei in 45 µL of 1x Tango Buffer and mix with 30 µL of tagmentation mix. The final concentration of Tn5 and DMF in final reaction is 0.8 µM and 16% (vol/vol), respectively. The ratio of nuclei suspension and tagmentation mix is 3 : 2 or 45 µL : 30 µL. Add nuclei into 1.5 mL DNA LoBind tube then add tagmentation mix and mix gently by pipetting 10 times. Briefly spin the tube on mini centrifuge at room temperature for 3 s and aliquot 75 µL of tagmentation reaction into 1.5 mL DNA LoBind tube. 
SECTION: Tagmentation
55. CRITICAL STEP: Set up 4 tubes of tagmentation reactions to have enough nuclei for Round 1 barcoding (8,000 nuclei/well x total 48 wells = 384,000 nuclei). Set up the reactions on ice. Do not incubate tagmentation reaction in large volume to make sure that nuclei are distributed evenly in the reaction not sitting on the bottom of the tube when incubating during tagmentation.
SECTION: Tagmentation
56. Place the tubes on thermomixer and incubate at 37 ºC for 30 min, 300 rpm.
30 min 

SECTION: Tagmentation
57. Before incubation is complete, prepare 1x PBS + 0.1% (wt/vol) BSA + RI (1 mL per 400 µL tagmented nuclei)  and keep on ice

1000 µL 
13.33 µL 
1.5 µL 
0.75 µL 
SECTION: Tagmentation
58. Remove the tubes from thermomixer and place on ice. Then pool tagmented nuclei of the same sample into the same tube.
SECTION: Tagmentation
59. Add 2.5x volume of 1x PBS + 0.1% (wt/vol) BSA + RI (1000 µL to 400 µL tagmented nuclei) to pooled tagmented nuclei and mix by pipetting gently 5 times and centrifuge at 900 g for 8 min, 4 ºC with swinging bucket rotor
SECTION: Tagmentation
60. During centrifugation, prepare 0.5x PBS + RI (1 mL per 1 million nuclei/cells) and keep on ice
500 µL 
500 µL 
2.5 µL 
1.25 µL 
SECTION: Tagmentation
61. CRITICAL STEP Aspirate supernatant and resuspend nuclei with 300 µL of 0.5x PBS + RI to have a minimum concentration of nuclei not lower than 1,000 nuclei/µL. If different numbers of nuclei in tagmentation are used, adjust suspension volume. Pipetette gently to resuspend
SECTION: Tagmentation
62. Count nuclei concentration with cell counter and dilute nuclei to 1,000 nuclei/µL with 0.5x PBS + RI.
SECTION: Accessible chromatin (AC) oligo ligation
63. Thaw Round 1 AC/RT oligo working plate on ice and spin the plate on swinging bucket at 200 g for 1 min, 4 ºC and leave the plate on ice. Note: Can leave in 4C at the beginning of Day 1 to thaw.
SECTION: Accessible chromatin (AC) oligo ligation
64. Prepare GLR-A mix following table below. CRITICAL STEP: Prepare GLR-A mix during washing tagmented nuclei, but add ATP, RNase inhibitor and T7 DNA Ligase just before ready to load into Round 1 barcoding plate
260 µL 
20.8 µL 
52 µL 
13 µL 
6.76 µL 
10.4 µL 
78 µL 
1.04 µL 

SECTION: Accessible chromatin (AC) oligo ligation
65. Add 8 µL of nuclei to each well (row A – D) with a multichannel pipette. 
SECTION: Accessible chromatin (AC) oligo ligation
66. Aliquot 73 µL of GLR-A into 6 tube-strip on PCR cool rack and add 8.5 µL of GLR-A mix to each well with a multichannel pipette. 
SECTION: Accessible chromatin (AC) oligo ligation
67. Seal and quick spin the plate at 160 g for 15 s, 4 ºC. Gently mix reaction mix, Round 1 barcoded oligos and nuclei 5 times (mixing volume 18 µL)
SECTION: Accessible chromatin (AC) oligo ligation
68. Seal and quick spin the plate at 160 g for 10 s, 4 ºC and incubate the plate on thermomixer at 25 ºC for 30 min, 300 rpm
30 min 

SECTION: Reverse transcription
69. Aliquot 20 µL of Maxima H Minus Reverse Transcriptase into 6 tubes of PCR strip tube
120 µL 

SECTION: Reverse transcription
70. Remove Round 1 barcoding plate from thermomixer, put on PCR cool rack, and add 2.2 µL of Maxima H Minus Reverse Transcriptase to each well with a multichannel pipette. CRITICAL STEP: To get accurate volumes, set the speed of multichannel pipette to be very slow for aspirating and dispensing as the enzyme is very viscous
SECTION: Reverse transcription
71. Seal and quick spin the plate at 160 g for 15 s, 4 ºC to bring enzyme to the bottom of the well and mix by gently pipetting 5 times (mixing volume 18 µL). Seal and quick spin the plate at 160 g for 10 s, 4ºC
SECTION: Reverse transcription
72. Incubate the plate on thermocycler using the program: 50ºC for 10 min, 3 cycles of (8ºC for 12 s, 15ºC for 45 s, 20ºC for 45 s, 30ºC 30 s, 42ºC for 2 min, 50ºC for 3 min), 50ºC for 5 min.
SECTION: Round 2 DNA barcoding
73. Before reverse transcription finishes, prepare 3 mL of 1x PBS + 0.1% BSA + RI

3000 µL 
40 µL 
4.5 µL 
2.25 µL 
SECTION: Round 2 DNA barcoding
74. Remove the plate from thermocycler and put on ice or PCR cool rack then pool all reactions into a chilled 25 mL reservoir and transfer pooled reaction into a chilled 5 mL DNA LoBind tube. CRITICAL STEP: Before pooling, pipette Round 1 barcoding plate 2 times to kick up nuclei from the bottom of the well and make sure that you transfer all reactions from the well by aspirating slowly and set pipette volume to 26 µL. This technique should be done for all pooling steps. Also keep reactions on ice all the time when handling samples or reaction mixes to prevent RNA degradation and preserve enzyme activity or temperature sensitive reagents like ATP.
SECTION: Round 2 DNA barcoding
75. Add 2.8 mL of 1x PBS + 0.1% (wt/vol) BSA + RI (2.5x volume) to rinse the basin and transfer buffer to the tube
SECTION: Round 2 DNA barcoding
76. Add 19.5 µL of 10% (vol/vol) Triton X-100 (final concentration of Triton X-100 is 0.05% (vol/vol)) and mix by inverting the tube 5 times before centrifuge at 900 g for 8 min, 4 ºC
19.5 µL 

SECTION: Round 2 DNA barcoding
77. Thaw Round 2 barcoded oligos working plate on ice and spin the plate at 200 g, for 1 min, 4 ºC and leave the plate on ice. Note: Can leave in 4C at the beginning of Day 1 to thaw.
SECTION: Round 2 DNA barcoding
78. Prepare 1x Buffer 3.1 as follows: 
210 µL 
21 µL 
1890 µL 

SECTION: Round 2 DNA barcoding
79. Prepare Ligation Mix as follows:
510 µL 
40.8 µL 
12.75 µL 
51 µL 
127.5 µL 
1338.8 µL 

SECTION: Round 2 DNA barcoding
80. Remove supernatant as much as possible (~20 µL left) and resuspend nuclei with 2.02 mL of 1x Buffer 3.1 and add 2.04 mL of Ligation mix and mix by pipetting 10 times
SECTION: Round 2 DNA barcoding
81. Transfer nuclei in ligation mix into 25 mL reservoir and add 40 µL of nuclei suspension into each well of Round 2 barcoding plate and mix gently by pipetting 5 times (mixing volume 45 µL)
SECTION: Round 2 DNA barcoding
82. Seal the plate with sealing film and quick spin the plate at 160 g for 10 s, 4 ºC
SECTION: Round 2 DNA barcoding
83. Incubate the plate on thermomixer at 37 ºC for 30 min, 300 rpm
30 min 

SECTION: Round 2 DNA barcoding
84. Prepare Round 2 blocking solution as follows:
47.52 µL 
300 µL 
852.5 µL 

SECTION: Round 2 DNA barcoding
85. Remove Round 2 DNA barcoding plate from thermomixer and quick spin at 160 g for 10 s, 4 ºC. Add 10 µL of Round 2 blocking solution to each well with multichannel pipette and mix by pipetting gently 5 times (mixing volume 55 µL)
SECTION: Round 2 DNA barcoding
86. Seal the plate with sealing film and quick spin at 160 g for 10 s, 4ºC then incubate the plate on thermomixer at 37 ºC for 30 min, 300 rpm
30 min 

SECTION: Round 3 DNA barcoding
87. Thaw Round 3 barcoded oligos working plate on ice, spin the plate on swinging bucket at 200 g for 1 min, 4 ºC and leave the plate on ice
SECTION: Round 3 DNA barcoding
88. Place Round 2 DNA barcoding plate on PCR cool rack and pool into 25 mL reservoir. Add 100 µL of T4 DNA Ligase (400 U/µL) into the basin with nuclei from Round 2 barcoding plate and mix well by gently pipetting 10 times and rock the basin from side-to-side 10 times
100 µL 

SECTION: Round 3 DNA barcoding
89. Add 50 µL of nuclei suspension to each well of Round 3 DNA barcoding plate and mix gently by pipetting 5 times (mixing volume 55 µL).
SECTION: Round 3 DNA barcoding
90. Seal the plate with sealing film and briefly spin the plate at 160 g for 10 s, 4ºC. Incubate the plate on thermomixer at 37 ºC for 30min, 300 rpm
30 min 

SECTION: Round 3 DNA barcoding
91. Prepare Round 3 blocking solution as follows:
41.4 µL 
600 µL 
1758.6 µL 

SECTION: Round 3 DNA barcoding
92. Remove Round 3 DNA barcoding plate from thermomixer and add 20 µL of Round 3 blocking solution to each well and gently mix by pipetting 3 times (mixing volume 75 µL)
SECTION: Round 3 DNA barcoding
93. Without incubation, pool the reaction into 25 mL reservoir placed on ice, transfer supernatant into 15 mL tube and centrifuge at 1,000 g for 8 min, 4ºC. OPTIONAL: EDTA in the reaction inhibits ligase activity, therefor there is no need to change the pipette tips when pooling Round 3 ligation reaction. Set pipetting volume 85 µL when pooling nuclei.
SECTION: Round 3 DNA barcoding
94. In parallel, prepare chilled wash buffer as follows. 
4000 µL 
40 µL 
10 µL

SECTION: Round 3 DNA barcoding
94.1. Also in parallel, thaw 2X Lysis buffer at 37C for ~15 minutes  if previously prepped. If not see step 6 for recipe.
SECTION: Round 3 DNA barcoding
95. Remove supernatant and add 4 mL of wash buffer. Pipette gently 5 times with p1000 pipette then centrifuge at 1,000 g for 8 min, 4 ºC
SECTION: Round 3 DNA barcoding
96. Carefully remove supernatant as much as possible and resuspend nuclei with 300 µL of 1x PBS + RI 
500 µL 
5 µL 
2.5 µL 
SECTION: Round 3 DNA barcoding
97. Count nuclei concentration with cell counter and aliquot nuclei at required number in each pool in 1.5 mL DNA LoBind tube and adjust total volume of nuclei to 50 µL with 1x PBS + RI. CRITICAL STEP: To sequence all nuclei, recommend using ≤20,000 cells per pool by splitting 50 µL of nuclei suspension into 6 - 12 tubes per plate.
SECTION: Nuclei lysis
98. Add 50 µL of 2x Lysis buffer and 10 µL of 20 mg/mL Proteinase K to each pool, mix well by gently vortexing and brief spin the tube down at room temperature for 10 s to collect all nuclei suspension to the bottom of the tube and incubate on thermomixer at 55 ºC for 2 h, 350 rpm to lyse nuclei and reverse crosslink formaldehyde fixation.
120 min 

SECTION: Nuclei lysis
99. Put nuclei lysate at -80 ºC to inactivate Proteinase K before continue to day 2 experiment. CRITICAL STEP: Lysis buffer tends to precipitate at room temperature or low temperature. Redissolve by incubating the tube at 37 ºC until it completely dissolves before adding to nuclei suspension. PAUSE POINT: Nuclear or cell lysate can be store at -80 ºC for up to 6 months before continue to day 2 experiment.
SECTION: Dynabeads MyOne streptavidin beads preparation
100. Prepare 3.5 mL of 1x B&W-T + RI for bead washing:
3500 µL 
5 µL 

SECTION: Dynabeads MyOne streptavidin beads preparation
101. Vortex the bottle of Dynabeads MyOne C1 thoroughly and aliquot required volume (44 µL/pool * number of pool) into 1.5 mL tube. Add 800 µL of 1x B&W-T + RI, mix by vortexing and pulse spin on mini centrifuge at room temperature for 3 s
44 µL 

SECTION: Dynabeads MyOne streptavidin beads preparation
102. Place the tubes onto the magnetic rack until liquid is clear; Remove supernatant with p1000 pipette
SECTION: Dynabeads MyOne streptavidin beads preparation
103. Resuspend the beads with 800 µL of 1x B&W-T + RI, vortex and pulse spin on mini centrifuge at room temperature for 3 s
SECTION: Dynabeads MyOne streptavidin beads preparation
104. Place the tubes onto the magnetic rack until solution is clear and remove supernatant with p1000 pipette
SECTION: Dynabeads MyOne streptavidin beads preparation
105. Repeat washes two more times (total of 3 washes)
SECTION: Dynabeads MyOne streptavidin beads preparation
106. Resuspend the beads with 100 µL of 2x B&W + RI per pool:
100 µL 
2 µL 

SECTION: cDNA/DNA capture
107. During bead preparation, take the tubes of lysate out of -80 ºC and place onto thermomixer set at 55 ºC for 2 min until lysate is completely thawed
SECTION: cDNA/DNA capture
108. Add 5 µL of 0.1 M PMSF (from -20 ºC) to each tube, pulse vortex for 10 s and pulse spin on mini centrifuge at room temperature for 5 s and incubate at room temperature for 10 min with no shaking
5 µL 
10 min 

SECTION: cDNA/DNA capture
109. Add 100 µL of streptavidin beads in 2x B&W + RI to each tube of lysate (no pipetting required) then agitate the tubes on mixer at room temperature for 1 h, 1,200 rpm. CRITICAL STEP: We recommend to use orbital shaker for microtubes. The speed of mixer can be adjusted as long as the beads do not settle on the bottom of the tube
60 min 

SECTION: cDNA/DNA capture
110. Pulse spin the tubes on mini centrifuge at room temperature for 5 s, place onto magnetic rack until solution is clear and remove supernatant with p200 pipette. CRITICAL STEP: Every time before placing the tube back to the magnet, quick spin the tubes on mini centrifuge for 3-5 s to collect all supernatant/lysate/buffer and beads to the bottom of the tube. Use p200 pipette to remove supernatant to avoid disturbing the beads and prevent bead loss.
SECTION: cDNA/DNA capture
111. Prepare 1 mL of 1x B&W-T + RI (750 uL per lysate) for bead washing as follows:
1000 µL 
1.4 µL 

SECTION: cDNA/DNA capture
112. Add 250 µL of 1 x B&W-T + RI and agitate the bead at room temperature for 5 min, 1,500 rpm to wash the beads
5 min 

SECTION: cDNA/DNA capture
113. Place the tube onto magnetic rack until solution is clear and remove supernatant. Continue to template switching oligo blocking on AC DNA immediately.
SECTION: Blocking template switching oligo
114. During working on Steps 109-110, prepare Nextera adapter 1 blocking solution per pool:
250 µL 
2.5 µL 
1 µL 

SECTION: Blocking template switching oligo
115. Add 250 µL of 6x SSC to each tube without bead suspension and wait until the supernatant is clear then remove supernatant with p200 pipette
250 µL 

SECTION: Blocking template switching oligo
116. Add 250 µL of Nextera adapter 1 blocking solution to each tube and agitate the tubes on mixer at room temperature for 1 min, 1,500 rpm then reduce the speed to 500 rpm for 14 min. CRITICAL STEP: Agitate the tubes at high speed 1,500 rpm for 1 min to make sure the beads are resuspended well, then shake gently at 500 rpm for 14 min to allow hybridization of Nextera adapter 1 blocker and Nextera adpter 1 on AC DNA and make sure that the beads do not settle.
15 min 

SECTION: Blocking template switching oligo
117. Place the tube onto magnetic rack until solution is clear and remove supernatant with p200 pipette.
SECTION: Blocking template switching oligo
118. Wash the beads twice with 1x B&W-T + RI as described above at room temperature for 5 min each round.
5 min 
5 min 

SECTION: Blocking template switching oligo
119. In parallel, prepare Tris-T + RI during second bead wash
250 µL 
2.5 µL 
0.63 µL 

SECTION: Blocking template switching oligo
120. Wash the beads with 250 µL of Tris-T + RI (5 min) the same way as 1x B&W-T + RI wash (at room temperature for 5 min, 1,500 rpm). In parallel, prepare GLR-B mix. CRITICAL STEP: If GLR-B mix is not ready, leave the beads in the tube with Tris-T + RI on ice until GLR-B mix is ready
5 min 

SECTION: Gap filling, ligation and complete reverse transcription
121. During washing, prepare GLR-B mix as follows:
50 µL 
4 µL 
20 µL 
10 µL 
5 µL 
2 µL 
12.5 µL 
2.5 µL 
89 µL 

SECTION: Gap filling, ligation and complete reverse transcription
122. Place the tubes onto magnetic rack until solution is clear, remove supernatant with p200 pipette and add 250 µL of nuclease-free water to each tube without bead suspension
250 µL 

SECTION: Gap filling, ligation and complete reverse transcription
123. Remove water and resuspend the beads with 195 µL of GLR-B mix by gently vortexing and quick spin the tubes on mini centrifuge for 3 s
SECTION: Gap filling, ligation and complete reverse transcription
124. Rotate the tubes in incubator at 37 ºC with slow speed for 15 min to allow for gap filling on AC DNA and ligate Nextera adapter 1 blocker to the AC DNA. OPTIONAL: For any step required rotator, thermomixer can be alternatively used as long as the tube is shaken gently and the beads do not settle.
15 min 

SECTION: Gap filling, ligation and complete reverse transcription
125. Remove the tubes from incubator and add each tube with 5 µL of 100 µM TSO and 5 µL of Maxima H Minus reverse transcriptase and mix well by gently vortexing. 
5 µL 
5 µL 

SECTION: Gap filling, ligation and complete reverse transcription
126.  Continue to incubate at room temperature for 30 min with slow rotation.
30 min 

SECTION: Gap filling, ligation and complete reverse transcription
127. Incubate at 42 ºC for 90 min with slow rotation.
90 min 

SECTION: Gap filling, ligation and complete reverse transcription
128. PAUSE POINT: The beads can be stored in Tris-T buffer at 4 ºC overnight before continuing to 1st PCR (Remove supernatant and replace with Tris-T buffer). However, we recommend to continue to the 1st PCR immediately if possible.
SECTION: First PCR, cDNA/DNA amplification
129. Before finishing 42C incubation, set up the first PCR mix to amplify both cDNA and accessible chromatin (AC) DNA and prepare PCR strip tubes with individual hinged cap (4 tubes per pool):
110 µL 
6.9 µL 
17.6 µL 
6.9 µL 
6.9 µL 
74.8 µL 

Note: 
Sequence of Hash_PCR: 	ACACTCTTTCCCTACACGACGCTCTTCCGATCT
SECTION: First PCR, cDNA/DNA amplification
130. After finishing incubation at 42 ºC for 90 min, place the tubes onto the magnetic rack until liquid is clear and remove supernatant with p200 pipette 
SECTION: First PCR, cDNA/DNA amplification
131. Add 250 µL of nuclease-free water to each tube without bead resuspension. Once liquid is clear, remove supernatant.
250 µL 

SECTION: First PCR, cDNA/DNA amplification
132. Resuspend the beads with 220 µL of first PCR mix, quick spin and aliquot 55 µL of bead suspension in PCR mix to each of 4 PCR strip tubes. Transfer all the leftover beads to 4 PCR tubes equally. CRITICAL STEP: Transfer the beads in PCR mix directly to the bottom of the tube, so there is no need to spin the tube before placing on thermocycler. Try to transfer PCR mix with the beads into PCR strip tubes as quick as possible to minimize bead settling before the reaction starts.
SECTION: First PCR, cDNA/DNA amplification
133. Place the tubes on thermocycler and run following program: 95 ºC for 3 min,  9 cycles of (98 ºC for 20 s, 58 ºC for 45 s, 72 ºC for 3 min), 72 ºC for 5 min, 4 ºC hold.  

SECTION: First PCR, cDNA/DNA amplification
134. PAUSE POINT PCR reaction can be stored at -20 ºC for a month or 4 ºC for a week.
SECTION: First PCR, cDNA/DNA amplification
135. Place strip tubes onto 0.2 mL magnetic rack until supernatant is clear and pool 1st round PCR products from 4 strip tubes of the same pool into 1.5 mL DNA LoBind tube
SECTION: First PCR, cDNA/DNA amplification
136. Vortex the tube and quick spin on mini centrifuge for 3 s before splitting PCR products into two parts (100 µL each), “AC” for chromatin accessibility (AC) library preparation and “R” for RNA library preparation. Note: AC and R in this step are the same PCR products but will be bead size-selected at different bead volume ratio.
SECTION: sn/scATAC libraries: purifiation and validation
137. Perform one round of 1.2x KAPA Pure Beads purification following manufacturer’s instructions by using 120 µL of KAPA Pure Beads with 100 µL of PCR products and elute with 40 µL of nuclease-free water
1. Binding
120 µL 8 min 
2. Washing
180 µL 30 s 
3. 2nd wash
180 µL 30 s 
4. Drying
1 min 37 °C 
5. Elution
 40 µL  10 min 37 °C 


SECTION: sn/scATAC libraries: purifiation and validation
138. Transfer eluent into new PCR strip tubes. The resultant is called AC-A.
SECTION: sn/scATAC libraries: purifiation and validation
139. Use 2 µL of AC-A product to determine DNA concentration with Qubit dsDNA HS assay kit following manufacturer’s instruction
SECTION: sn/scATAC libraries: purifiation and validation
140. Verify ~10 ng of AC-A in 6% TBE gel and run in 1x TBE buffer at 250 volts for 23 min with 0.5 µL of Low DNA Mass Ladder as reference
SECTION: sn/scATAC libraries: 2nd PCR and library preparation
141. In PCR strip tubes, use 5 ng of AC-A as template for enrichment of AC DNA over cDNA and adjust volume of template to 10 µL with nuclease-free water and quick spin down the tubes. CRITICAL STEP: If the concentration of PCR product is higher than 5 ng/µL, it tends to have high error to pipette the volume smaller than 1 µL. We recommend to aliquot AC-A, dilute into 0.5 ng/µL in total 30-50 µL and use 10 µL as template for AC 2nd PCR.
10 µL 

SECTION: sn/scATAC libraries: 2nd PCR and library preparation
142. Prepare PCR mix as follows:
25 µL 
2.5 µL 
2.5 µL 
7.5 µL 

SECTION: sn/scATAC libraries: 2nd PCR and library preparation
143. Add 37.5 µL of PCR mix and 2.5 µL of 10 µM Ad1_N50X (attached tables) into the tube with AC-A template
2.5 µL 

SECTION: sn/scATAC libraries: 2nd PCR and library preparation
144. Mix by gently vortexing, quick spin PCR tubes on mini centrifuge at room temperature for 3 s and run qPCR on real-time PCR machine: 95 ºC for 3 min,  12 (or fewer) cycles of (98 ºC for 20 s, 58 ºC for 45 s, 72 ºC for 1 min), 72 ºC for 5 min, 4 ºC hold.  

SECTION: sn/scATAC libraries: 2nd PCR and library preparation
145. PAUSE POINT: PCR reaction can be stored at -20 ºC for a month or 4 ºC for a week.
SECTION: sn/scATAC libraries: 2nd PCR and library preparation
146. Purify PCR product with DNA Clean & Concentrator following manufacturer's instructions and elute with 40 µL of DNA Elution Buffer. Resultant is called AC-B
SECTION: sn/scATAC libraries: 2nd PCR and library preparation
147. Use 2 µL of AC-B to determine DNA concentration with Qubit dsDNA HS assay kit following manufacturing’s instruction
SECTION: sn/scATAC libraries: 2nd PCR and library preparation
148. Verify ~10 ng of AC-B in 6% TBE gel and run in 1x TBE buffer at 250 volts for 23 min with 0.5 µL of Low DNA Mass Ladder as a reference in separate lane. CRITICAL STEP: We expect to see nucleosome pattern with larger size (~125 bp larger) compare to standard ATAC-seq due to the presence of cell barcodes and linker sequences
SECTION: sn/scATAC libraries: 2nd PCR and library preparation
149. Pool equimolar ratio of AC-B libraries (~200-250 ng/pool) and perform PAGE size-selection at the size between 300-1,000 bp.
SECTION: sn/scATAC libraries: 2nd PCR and library preparation
150. Use 2 µL of AC sequencing libraries to determine for DNA concentration with Qubit dsDNA HS assay
SECTION: sn/scATAC libraries: 2nd PCR and library preparation
151. Verify ~5 -10 ng of AC sequencing libraries in 6% TBE gel and run in 1x TBE buffer at 250 volts for 23 min or by TapeStation
SECTION: sn/scATAC libraries: Quality validation by MiSeq sequencing
152. Validate AC sequencing libraries with MiSeq sequencing using v2 reagent kit by loading at 20 pm based on Qubit dsDNA HS quantification with at least 5% PhiX spike in following Illumina  loading guide. The sequencing configuration is 75 cycles for read 1, 94 cycles for index 1, 8 cycles for index 2, and 75 cycles for read 2.
 

SECTION: sn/scATAC libraries: Quality validation by MiSeq sequencing
153. Mix 1:1 ratio of "SN2-AC R1" sequencing primer pairs for sequencing read 1 on Illumina workflow A (HiSeq 2500, MiSeq, NovaSeq 6000 platforms).
3 µL 
3 µL 
594 µL 

SECTION: sn/scATAC libraries: Quality validation by MiSeq sequencing
154. Mix 1:1 ratio of "SN2-AC R2" sequencing primer pairs for sequencing index 1 on Illumina workflow A (HiSeq 2500, MiSeq, NovaSeq 6000 platforms).
3 µL 
3 µL 
594 µL 

SECTION: sn/scATAC libraries: Quality validation by MiSeq sequencing
155. Mix 1:1 ratio of "SN2-AC R4" sequencing primer pairs for sequencing read 2 on Illumina workflow A (HiSeq 2500, MiSeq, NovaSeq 6000 platforms).
3 µL 
3 µL 
594 µL 
SECTION: sn/scRNA libraries: purification and validation
156. Perform 1 round of 0.8x KAPA Pure Beads purification following manufacturer’s instructions by mixing 100 µL of 1st PCR product + with 80 µL of KAPA Pure Beads and elute with 40 µL of nuclease-free water. Resultant is called R-A.
1. Binding
80 µL 8 min 
2. Washing
180 µL 30 s 
3. 2nd wash
180 µL 30 s 
4. Drying
1 min 37 °C 
5. Elution
40 µL 10 min   

SECTION: sn/scRNA libraries: purification and validation
157. Use 2 µL of R-A to determine DNA concentration with Qubit dsDNA HS assay following manufacturer’s instructions
SECTION: sn/scRNA libraries: purification and validation
158. Verify 5-10 ng of R-A in 6% TBE gel and run in 1x TBE buffer at 250 volts for 23 min with 0.5 µL of Low DNA Mass Ladder as a reference
SECTION: sn/scRNA libraries: 2nd PCR, purification, and validation
159. In PCR strip tubes, use 5 ng of R-A as template for enrichment of cDNA over AC DNA and adjust the volume of template to 10 µL with nuclease-free water and quick spin down the tubes. CRITICAL STEP: If the concentration of PCR product is higher than 5 ng/µL, it tends to have high error to pipette the volume smaller than 1 µL. We recommend to aliquot R-A, dilute into 0.5 ng/µL in total 30-50 µL and use 10 µL as template for RNA 2nd PCR
SECTION: sn/scRNA libraries: 2nd PCR, purification, and validation
160. Prepare PCR mix for second round amplification of cDNA as follows:
25 µL 
2 µL 
2 µL 
2.5 µL 
8.5 µL 

SECTION: sn/scRNA libraries: 2nd PCR, purification, and validation
161. Add 40 µL of PCR mix to each tube, gently vortex and quick spin PCR tubes on mini centrifuge at room temperature for 3 s
SECTION: sn/scRNA libraries: 2nd PCR, purification, and validation
162. Run the reactions on real-time PCR using following program: 95 ºC for 3 min,  12 (or fewer) cycles of (98 ºC for 20 s, 67 ºC for 45 s, 72 ºC for 3 min), 72 ºC for 5 min, 4 ºC hold. 

SECTION: sn/scRNA libraries: 2nd PCR, purification, and validation
163. PAUSE POINT PCR reaction can be stored at -20 ºC for a month or 4 ºC for a week.
SECTION: sn/scRNA libraries: 2nd PCR, purification, and validation
164. Purify PCR products with 1 round of 0.8x KAPA Pure Beads and elute with 40 µL of nuclease-free water. The resultant purified DNA is called R-B
1. Binding
40 µL 8 min 
2. Washing
180 µL 30 s 
3. 2nd wash
180 µL 30 s 
4. Drying
1 min 37 °C 
5. Elution
40 µL 10 min 37 °C 

SECTION: sn/scRNA libraries: 2nd PCR, purification, and validation
165. Determine DNA concentration with Qubit dsDNA HS assay using 2 µL of R-B
SECTION: sn/scRNA libraries: 2nd PCR, purification, and validation
166. Verify ~5-10 ng of R-B in 6% TBE gel by running at 250 volts for 23 min with 0.5 µL of Low DNA Mass Ladder as a reference. CRITICAL STEP: If the smear of R-B is between 375 bp and above, continue to cDNA tagmentation. If there is strong smear smaller than 375 bp, repeat another round of 0.8x KAKA Pure Beads and elute with 40 µL of nuclease-free water.
SECTION: sn/scRNA libraries: cDNA tagmentation and library preparation
174. Mix ME and Nextera Adapter 1 (Ad1) oligos 1:1 for final 50 uM each. Briefly vortex and quick spin the tube. Anneal transposons on thermocycler using following program: 95 ºC 5 min, slowly cool down to 65 ºC (0.1 ºC/s), 65 ºC 5 min, slowly cool down to 4 ºC (0.1 ºC/s) and hold at 4 ºC.
SECTION: sn/scRNA libraries: cDNA tagmentation and library preparation
175. Load Tn5 (In-house, 11.32 uM) 
20 µL 
6.8 µL 

SECTION: sn/scRNA libraries: cDNA tagmentation and library preparation
176. Incubate 1 hour at room temperate with gentle shaking.
60 min 

SECTION: sn/scRNA libraries: cDNA tagmentation and library preparation
177. Prepare Tagmentation mix as follows in 1.5 mL tube and aliquot 18 µL per pool into 0.2 mL PCR strip tubes with hinged cap.
2 µL 
2 µL 
12 µL 
2 µL 

SECTION: sn/scRNA libraries: cDNA tagmentation and library preparation
178. Transfer 2 µL of diluted cDNA (total 10-20 ng) with multichannel pipette into tagmentation mix, mix by pipetting 10 times, gently vortex and quick spin on mini centrifuge at room temperature for 5 s
SECTION: sn/scRNA libraries: cDNA tagmentation and library preparation
179. Place the tubes on thermocycler that set at 55 ºC for 7 min with the heated lid
7 min 

SECTION: sn/scRNA libraries: cDNA tagmentation and library preparation
180. Remove the tube from thermocycler and stop reaction by adding 5 µL of 0.2 % (wt/vol) SDS, mix by pipetting 5 times, gently spin down the tubes on mini centrifuge for 5 s and incubate at room temperature for 5 min
5 µL 
5 min 

SECTION: sn/scRNA libraries: cDNA tagmentation and library preparation
181. Set up Tagmentation PCR mix during incubation
25 µL 
2.5 µL 
2.5 µL 
7.5 µL 


SECTION: sn/scRNA libraries: cDNA tagmentation and library preparation
182. Add  2.5 SPLiT_N7XX (attached tables) primers to 10 uL tagmented cDNA, then 37.5 of Tagmentation PCR mix respectively, mix well and gently spin down the tube on mini centrifuge for 3 s
2.5 µL 
10 µL 
37.5 µL 

SECTION: sn/scRNA libraries: cDNA tagmentation and library preparation
183. Run PCR on thermocycler using program follows: 72 ºC for 3 min, 98 ºC for 30 s,  9-12 cycles of (98 ºC for 30 s, 63 ºC for 30 s, 72 ºC for 45 s), 72 ºC for 3 min, 4 ºC hold. Terminate the reaction when it reaches mid-exponential growth.
SECTION: sn/scRNA libraries: cDNA tagmentation and library preparation
184. Purify PCR products at least two rounds of 0.7x KAPA Pure beads to make sure that no adapter dimers are leftover in the sequencing libraries. Elute the last round of bead purified RNA libraries with 30 µL of nuclease-free water. CRITICAL STEP: Adapter dimers can cluster more efficiently than regular sequencing libraries and is more sensitive on the patterned flow cell such as NovaSeq S4. This can lead to the reduction of sequencing output and sequencing quality
1. Binding
35 µL 8 min 
2. Washing
180 µL 30 s 
3. 2nd wash
180 µL 30 s 
4. Drying
1 min 37 °C 
5. Elution
50 µL 10 min 37 °C 
6. Binding
35 µL 8 min 
7. Washing
180 µL 30 s 
8. 2nd wash
180 µL 30 s 
9. Drying
1 min 37 °C 
10. Elution
30 µL 10 min 37 °C 


SECTION: sn/scRNA libraries: cDNA tagmentation and library preparation
185. Use 1 µL of RNA sequencing libraries to determine for DNA concentration with Qubit dsDNA HS assay
SECTION: sn/scRNA libraries: cDNA tagmentation and library preparation
186. Verify ~ 5 ng of RNA libraries in 6% TBE gel at 250 volts for 23 min with 0.5 µL of Low DNA Mass Ladder as a reference
SECTION: sn/scRNA and hash libraries: Quality validation by MiSeq sequencing
187. Validate sequencing libraries with MiSeq sequencing using v3 reagent kit by loading at 22 pm based on Qubit dsDNA HS quantification with at least 5% PhiX spike.  The sequencing configuration is 70 cycles for read 1, 6 cycles for index, and 102 cycles for read 2.a

 

SECTION: sn/scRNA and hash libraries: Quality validation by MiSeq sequencing
188. Mix 1:1 ratio of "SN2-R R1" sequencing primer pairs for sequencing read 1 on Illumina workflow A (HiSeq 2500, MiSeq, NovaSeq 6000 platforms).
3 µL 
3 µL (Hash libraries uses the same Read 1 sequencing primer as PhiX)
594 µL 
SECTION: sn/scRNA and hash libraries: Quality validation by MiSeq sequencing
189. Mix 1:1 ratio of "SN2-R Index1" sequencing primer pairs for sequencing index 1 on Illumina workflow A (HiSeq 2500, MiSeq, NovaSeq 6000 platforms).
3 µL 
3 µL 
594 µL 
SECTION: sn/scRNA and hash libraries: Quality validation by MiSeq sequencing
190. Mix 1:1 ratio of "SN2-R R2" sequencing primer pairs for sequencing read 2 on Illumina workflow A (HiSeq 2500, MiSeq, NovaSeq 6000 platforms).
3 µL 
3 µL 
594 µL 
SECTION: Reagent setup
11. Samples hashing barcodes preparation. Resuspend oligonucleotides barcodes with nuclease-free water to 10 pM final concentration and make 20 uL aliquots of each oligo in 0.2 mL PCR tubes.  

 




SECTION: Nuclei isolation and fixation
41. To each tube of 0.5 mL volumes of nuclei suspensions, add 15 µL of hashing oligos. Make a note of which oligo was added to which sample. Mix briefly, centrifuge at 500 g for 8 minutes at 4C, remove 400 µL of supernatant. Then pool up to 8 barcoded samples 
SECTION: Hash libraries: 2nd PCR, purification, and validation
167. In PCR strip tubes, use 5 ng of R-A as template for enrichment of hashing libraries and adjust the volume of template to 10 µL with nuclease-free water and quick spin down the tubes. CRITICAL STEP: If the concentration of PCR product is higher than 5 ng/µL, it tends to have high error to pipette the volume smaller than 1 µL. We recommend to aliquot R-A, dilute into 0.5 ng/µL in total 30-50 µL and use 10 µL as template for 2nd PCR
SECTION: Hash libraries: 2nd PCR, purification, and validation
168. Set up 2nd PCR mix during incubation
25 µL 
2.5 µL 
2.5 µL 
7.5 µL 

Sequences of HASH_L0X (pick one of L01-3):
HASH_L01: AATGATACGGCGACCACCGAGATCTACACGTAACATGCGACACTCTTTCCCTACACGACG
HASH_L02: AATGATACGGCGACCACCGAGATCTACACGTGGATCAAAACACTCTTTCCCTACACGACG
HASH_L03: AATGATACGGCGACCACCGAGATCTACACCACTACGAAAACACTCTTTCCCTACACGACG
SECTION: Hash libraries: 2nd PCR, purification, and validation
169. Add 37.5 µL of PCR mix and 2.5 µL of 10 µM SPLiT_N7XX (attached tables) primers into the tube with R-A template
2.5 µL 
SECTION: Hash libraries: 2nd PCR, purification, and validation
170. Run PCR on thermocycler using program follows: 72 ºC for 3 min, 98 ºC for 30 s,  9-12 cycles of (98 ºC for 30 s, 63 ºC for 30 s, 72 ºC for 45 s), 72 ºC for 3 min, 4 ºC hold. Terminate the reaction when it reaches mid-exponential growth.
SECTION: Hash libraries: 2nd PCR, purification, and validation
171. Pool all reactions together, purify an aliquot of 50 uL with 1X beads. Elute purified hash libraries with 30 µL of nuclease-free water. 
1. Binding
50 µL 8 min 
2. Washing
180 µL 30 s 
3. 2nd wash
180 µL 30 s 
4. Drying
1 min 37 °C 
5. Elution
30 µL 10 min 37 °C 
SECTION: Hash libraries: 2nd PCR, purification, and validation
172. Use 1 µL of hash sequencing libraries to determine for DNA concentration with Qubit dsDNA HS assay
SECTION: Hash libraries: 2nd PCR, purification, and validation
173. Verify ~ 5 ng of hash libraries in 6% TBE gel at 250 volts for 23 min with 0.5 µL of Low DNA Mass Ladder as a reference.