TITLE: scNT-seq2: single-cell metabolically labelled new RNA tagging sequencing for time-resolved analysis of gene expression in single cells
AUTHORS: Qi Qiu, Fan Li, Dongming Liang, William Gao, Hao Wu

[DESCRIPTION]
Single-cell metabolically labeled new RNA tagging sequencing (scNT-Seq) is a droplet microfluidics-based, time-resolved RNA-seq method for joint profiling of newly synthesized (“new”) and pre-existing (“old”) RNAs from the same cell by marking new transcripts with T-to-C substitutions via chemical conversion of 4-thiouridine (4sU) in new RNAs to cytidine analogs (Qiu et al., Nature Methods 17.10 (2020): 991-1001. PMID: 32868927). Here, we described the updated scNT-Seq2 method in which an optimized second-strand synthesis reaction is employed to increase the mapping rate and library complexity.  This step-by-step protocol describes metabolic labeling, sample preparation, and scNT-Seq2 library preparation for Illumina sequencing.

[STEPS]
SECTION: Metabolic labeling
1. Prepare a 1 M stock solution of 4-thiouridine (4sU) by dissolving the powder in DMSO.
 

SECTION: Prepare cell suspension
3. After metabolic labeling, cells were rinsed once with DPBS.
SECTION: Metabolic labeling
2. For metabolic labeling, the medium was replaced with fresh medium supplemented with nontoxic concentrations of 4sU (i.e. 100 or 200 μM).
 

SECTION: Prepare cell suspension
4. Add 0.5 mL Accutase to each well of the 6-well plate and incubate at 37 C for about 5 min.
SECTION: Prepare cell suspension
9. Dilute the cell with DPBS (containing 0.01% BSA + 0.5%RNase-Inhibitor) to 100 cells/ μL (1 X 105 cells/mL).
SECTION: Prepare cell suspension
8. Count the cell number with Countess II.
SECTION: Prepare cell suspension
7. Resuspend the cell pellet with 1 mL of DPBS (containing 0.01% BSA + 0.5%RNase-Inhibitor).
SECTION: Prepare cell suspension
5. Add 1 mL culture medium to neutralize the Accutase.

SECTION: Prepare cell suspension
6. Collect cell suspension in the 15 mL tube, spin down cells at 350 g, 3min.
SECTION: Cell and beads co-encapsulation
10. Prepare lysis buffer:

 

SECTION: Cell and beads co-encapsulation
11. Prepare barcoded beads:
Wash beads once with 30 mL of 100% ethanol and twice with 30 mL of TE-TW (10 mM Tris-HCl pH 8.0, 1 mM EDTA and 0.01% Tween-20). Pass the beads through a 100 μm cell strainer and count the number of beads. Resuspend the beads at 120 beads/μL concentration in 1.5 mL lysis buffer for each run (to profile 1,000~2,000 cells). Transfer 1.5 mL of bead suspension into a 3 mL Luer lock syringe. Make one more mL of lysis buffer to wash the syringe and magnetic disc.
SECTION: Cell and beads co-encapsulation
12. Draw up 7 mL of droplet generation oil (Bio-Rad) into a 10-mL Luer-lock syringe.
SECTION: Cell and beads co-encapsulation
13. Connect 3 syringes (containing cells, beads, and oil, respectively) to the Aquapel-coated
PDMS Microfluidic device (μFluidix) with the following flow rate setting:

 

SECTION: Cell and beads co-encapsulation
14. Start the run in the following order: cells → beads → oil.
SECTION: Cell and beads co-encapsulation
15. When the flow of droplets stabilizes, collect ~20 μL of aqueous flow to examine the droplet quality. Check whether the droplet size is uniform and estimate the percentage of bead doublets (the doublet rate should be less than 5%).
SECTION: Cell and beads co-encapsulation
16. Once confirming the droplet quality, collect 1.2-1.3 mL of droplets into a 50 mL conical tube (target 1,000~2,000 cells for each sample).
SECTION: Droplet breakage
17. Remove the oil layer from the bottom of the 50 mL tube.
SECTION: Droplet breakage
18. Add 30 mL of room temperature 6X SSC into the tube.
SECTION: Droplet breakage
24. Spin at 1,000x g for 1 min. Remove the supernatant. Wash beads once with 1 mL of 6X SSC.
SECTION: Droplet breakage
23. The beads are now pelleted to the bottom of the tube. Carefully remove all but ~1 mL of liquid. Resuspend the beads with the remaining liquid and transfer them to a 1.5 mL Low Binding tube.
SECTION: Droplet breakage
22. Spin at 1,000x g for 1 min to pellet the beads.
SECTION: Droplet breakage
21. Add 25 mL of 6X SSC to kick up the beads into thesolution again. Transfer and combine the supernatant.
SECTION: Droplet breakage
19. Add 1 mL of Perfluorooctanol (PFO) into the tube in a fume hood. Shake by hand to break the droplets (3-4 forceful vertical shakes, the shakes should be long distance -- from head to leg) Spin at 1,000x g for 1 min.
SECTION: Droplet breakage
20. Carefully remove the supernatant on top and then add 25 mL of 6X SSC to kick up the beads into the solution. Wait a few seconds to allow the majority of the oil to sink to the bottom. Transfer the supernatant to a new 50 mL tube.
SECTION: Chemical conversion of mRNAs on beads
25. Wash beads once with 450 μL reaction buffer (without TFEA and NaIO4).
SECTION: Chemical conversion of mRNAs on beads
26. Incubate beads in 474 μL Reaction-mix + 26 μL NaIO4 at 45 C for 1 hr with rotation. 

 

SECTION: Chemical conversion of mRNAs on beads
27. Wash the beads once with 0.7 mL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5).
SECTION: Chemical conversion of mRNAs on beads
28. Incubate the beads in 0.7 mL Reducing Buffer at 37 C for 30 min with rotation. Add 2% RNase inhibitor (20 μL/1 mL) to the reducing buffer before use.

 

SECTION: Reverse transcription
29. Prepare RT mix:

 


SECTION: Reverse transcription
32. Add 220 μL of RT mix to the beads.
SECTION: Reverse transcription
33. Incubate beads at room temperature for 30 min with rotation, then 120 min at 42 C with rotation.
SECTION: Reverse transcription
34. Wash beads once with 1 mL TE-SDS (10 mM Tris-HCl pH 8.0, 1 mM EDTA, and 0.5% SDS), twice with 1 mL TE-TW, then store the beads at 4°C over-night (in TE-TW buffer). [This is a Stop point. Store at 4C overnight (in TE-Tw buffer).]
SECTION: Exonuclease I treatment
35. Prepare Exonuclease mix (makes 200 μL):

 

SECTION: Exonuclease I treatment
36. Wash beads once with 1 mL 10mM Tris-HCl pH 8.0, re-suspend in 190 μL of exonuclease mix.
SECTION: Exonuclease I treatment
37. Incubate beads at 37 C for 45 min with rotation.
SECTION: Exonuclease I treatment
38. Wash beads once with 1 mL TE-SDS, and twice with 1 mL TE-TW. [This is a Stop point. Store at 4C overnight (in TE-Tw buffer).]
SECTION: Run 1st round of TSO-PCR using 6,000-beads as templates
39. Wash beads once with 1 mL H2O. Spin at 1,000x g for 1 min.
SECTION: Run 1st round of TSO-PCR using 6,000-beads as templates
40. Remove supernatant and re-suspend the beads with 1 mL of H2O. Quickly transfer 2 μL of beads into a well of a 96-well plate (containing 198 μL of H2O) and count the number of beads. Repeat bead counting three times and take the average.
SECTION: Run 1st round of TSO-PCR using 6,000-beads as templates
41. Transfer an aliquot of 6,000 beads (corresponding to ~100 cells) into a PCR tube. Spin down and remove the supernatant, then re-suspend the beads with 50 μL PCR mix:

 

SECTION: Run 1st round of TSO-PCR using 6,000-beads as templates
42. Run 1st round of TSO-PCR. PCR program: 
Very important: run 1st round of TSO-PCR to determine the exact number of amplification cycles. Over-amplification of the cDNA library will lead to fewer detected nuclei in the end).

   95 C for 3 minutes
4 cycles of:
   98 C for 20 seconds
   65 C for 45 s
   72C for 3 min
9 cycles of:
   98 C for 20 s
   67 C for 20 s
   72 C for 3 min
Then:
   72 C for 5 min
   4 C forever
SECTION: Run 1st round of TSO-PCR using 6,000-beads as templates
43. Purify PCR products once with 0.7X (35 μL) SPRI beads once and elute in 15 μL Elution buffer.
SECTION: Run 1st round of TSO-PCR using 6,000-beads as templates
44. Measure the concentration of PCR products by Qubit.
SECTION: Run 1st round of TSO-PCR using 6,000-beads as templates
45. Perform real-time PCR to determine the additional number of PCR cycles needed for optimal cDNA amplification.

 

SECTION: Run 1st round of TSO-PCR using 6,000-beads as templates
46. Run real-time PCR with the following program, and determine the optimal PCR cycle number
   95 C for 3 min
25 cycles of:
   95 C for 15 s
   63 C for 30 s
   72 C for 30 s
SECTION: Large-scale TSO-PCR
56. Combine the PCR product for a given sample into a 1.5 mL Low Binding tube and purify twice with SPRI-select beads (0.6X Volume for the 1st & 0.7X Volume for the 2nd purification).
SECTION: Large-scale TSO-PCR
58. Quantify the cDNA library by Qubit and run the bioanalyzer to check the average fragment size of the purified cDNA library (the expected average size of cDNA library is 800-1,500 bp).
SECTION: Tagmentation (Nextera® XT DNA Sample Preparation kit)
59. Preheat the thermocycler to 55 C. For each sample, take out 1 ng of purified cDNA with H2O in a total volume of 5 μL to a PCR tube.
SECTION: Tagmentation (Nextera® XT DNA Sample Preparation kit)
60. Add 10 μL of Nextra TD buffer and 5 μL of Amplicon Tagmentation enzyme to each reaction. Mix by pipetting ~5 times.
SECTION: Tagmentation (Nextera® XT DNA Sample Preparation kit)
61. Incubate at 55 C for 5 min.
SECTION: Tagmentation (Nextera® XT DNA Sample Preparation kit)
62. Add 5 μL of Neutralization Buffer to each reaction. Mix by pipetting ~5 times. Spin down and incubate at room temperature for 5 min.
SECTION: Tagmentation (Nextera® XT DNA Sample Preparation kit)
63. Add to each PCR tube in the following order:

 

SECTION: Tagmentation (Nextera® XT DNA Sample Preparation kit)
64. PCR program:
   95 C for 30 s
12 cycles of:
   95 C for 10 s
   55 C for 30 s
   72 C for 30 s
Then:
   72 C for 5 min
   4 C forever
SECTION: Tagmentation (Nextera® XT DNA Sample Preparation kit)
65. Purify PCR product twice with 0.6X SPRI beads. Elute the cDNA in 12 μL H2O.
SECTION: Tagmentation (Nextera® XT DNA Sample Preparation kit)
66. Quantify the concentration of cDNA library by Qubit and check the average fragment size of the purified cDNA library by Bioanalyzer (the expected fragment size is 500-700 bp).
SECTION: Sequencing
67. Dilute the library to 2 nM and pool the libraries according to the estimated cell numbers (100 cells/6000 beads).
SECTION: Large-scale TSO-PCR
55. After determining the optimal PCR cycle number (usually an additional 0-4 cycles), perform large-scale TSO-PCR with the remaining beads. Wash the remaining beads twice with 1 mL H2O. Apportion 6,000 beads for each PCR reaction. Spin down and remove the supernatant, then resuspend the beads with 50 μL PCR mix. PCR program:

   95 C for 3 min
4 cycles of:
   98 C for 20 s
   65 C for 45 s
   72 C for 3 min
X plus additional cycles of:
   98 C for 20 s
    67 C for 20 s
   72 C for 3 min
Then:
   72 C for 5 min
   4 C forever
SECTION: Second strand synthesis
47. Prepare second strand synthesis mix:
 
 
 
SECTION: Second strand synthesis
48. After aspiration of TE-TW buffer, resuspend beads in 500 μL 0.1 M NaOH.
SECTION: Second strand synthesis
49. Incubate beads at room temperature for 5 min with rotation.
SECTION: Second strand synthesis
50. Add 500 μL 0.2M Tris-HCl (pH 7.5) to neutralize the solution. 
SECTION: Second strand synthesis
51. Wash beads once with TE-TW and once with 10 mM Tris-HCl (pH 8.0).
SECTION: Second strand synthesis
52. Add 200 μL second strand synthesis mix to the beads. [Add Bst3 enzyme right before the reaction.]
SECTION: Second strand synthesis
53. Incubate beads at 15 min at 60 C with rotation.
SECTION: Second strand synthesis
54. Wash beads once with 1 mL TE-SDS, and twice with 1 mL TE-TW and twice with H2O.
SECTION: Reverse transcription
30. Wash the beads once with 1 mL Tris-HCl buffer (10 mM, pH 7.5).
SECTION: Reverse transcription
31. Wash the beads once with 0.3 mL 2X RT buffer.
SECTION: Large-scale TSO-PCR
57. Elute the cDNA with 40 μL Low-EDTA TE.