scNT-seq2: single-cell metabolically labelled new RNA tagging sequencing for time-resolved analysis of gene expression in single cells

Prepare a 1 M stock solution of 4-thiouridine (4sU) by dissolving the powder in DMSO.

For metabolic labeling, the medium was replaced with fresh medium supplemented with nontoxic concentrations of 4sU (i.e. 100 or 200 μM).

After metabolic labeling, cells were rinsed once with DPBS.

Add 0.5 mL Accutase to each well of the 6-well plate and incubate at 37 C for about 5 min.

Add 1 mL culture medium to neutralize the Accutase.

Collect cell suspension in the 15 mL tube, spin down cells at 350 g, 3min.

Resuspend the cell pellet with 1 mL of DPBS (containing 0.01% BSA + 0.5%RNase-Inhibitor).

Count the cell number with Countess II.

Dilute the cell with DPBS (containing 0.01% BSA + 0.5%RNase-Inhibitor) to 100 cells/ μL (1 X 10⁵ cells/mL).

Prepare lysis buffer:

A	B	C
Reagents	Vol.(μL)	Final Concentration
H2O	400
20% Ficoll PM-400	300	6%
20% Sarkosyl	10	0.2%
0.5 M EDTA	40	20 mM
1.0 M Tris-HCl, pH 7.5	200	200 mM
1.0 M DTT (add freshly)	50	50 mM

Lysis buffer master mix

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.

Draw up 7 mL of droplet generation oil (Bio-Rad) into a 10-mL Luer-lock syringe.

Connect 3 syringes (containing cells, beads, and oil, respectively) to the Aquapel-coated

PDMS Microfluidic device (μFluidix) with the following flow rate setting:

A	B
Syringe Content	Flow Rate (μL/hr)
Oil	15000
Cells	4000
Beads	4000

Start the run in the following order: cells → beads → oil.

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%).

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).

Remove the oil layer from the bottom of the 50 mL tube.

Add 30 mL of room temperature 6X SSC into the tube.

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.

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.

Add 25 mL of 6X SSC to kick up the beads into thesolution again. Transfer and combine the supernatant.

Spin at 1,000x g for 1 min to pellet the beads.

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.

Spin at 1,000x g for 1 min. Remove the supernatant. Wash beads once with 1 mL of 6X SSC.

Wash beads once with 450 μL reaction buffer (without TFEA and NaIO₄).

Incubate beads in 474 μL Reaction-mix + 26 μL NaIO₄ at 45 C for 1 hr with rotation.

A	B
Reagents	Volume (μL)
3 M sodium acetate (pH 5.2)	8
0.5 M EDTA (pH 8.0)	2
H2O	214
TFEA	13
Sub-total	237
192 mM NaIO4	13

Master mix for chemical conversion

Wash the beads once with 0.7 mL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5).

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.

A	B
Reagents	For 1mL (μL)
1 M Tris-HCl (pH7.5)	10
1M DTT	10
5M NaCl	20
0.5M EDTA	2
RNase Inhibitor	20
DEPC-H2O	958

Reducing Buffer

Prepare RT mix:

A	B
Reagents	Vol. (μL)
H2O	80
Maxima 5X RT buffer	40
20% Ficoll PM-400	40
10 mM dNTPs	20
100 μM Template Switch Oligo	5
RNase Inhibitor	5
Maxima H Minus Reverse Transcriptase	10

Wash the beads once with 1 mL Tris-HCl buffer (10 mM, pH 7.5).

Wash the beads once with 0.3 mL 2X RT buffer.

Add 220 μL of RT mix to the beads.

Incubate beads at room temperature for 30 min with rotation, then 120 min at 42 C with rotation.

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).]

Prepare Exonuclease mix (makes 200 μL):

A	B
Reagents	Vol. (μL)
10X Exonuclease I buffer	20
H2O	170
Exonuclease I	10

Wash beads once with 1 mL 10mM Tris-HCl pH 8.0, re-suspend in 190 μL of exonuclease mix.

Incubate beads at 37 C for 45 min with rotation.

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).]

Wash beads once with 1 mL H₂O. Spin at 1,000x g for 1 min.

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.

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:

A	B
Reagents	Vol. (μL)
KAPA HiFi HS Readymix	25
100 μM TSO-PCR primer	0.4
H2O	24.6

TSO-PCR master mix

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

Purify PCR products once with 0.7X (35 μL) SPRI beads once and elute in 15 μL Elution buffer.

Measure the concentration of PCR products by Qubit.

Perform real-time PCR to determine the additional number of PCR cycles needed for optimal cDNA amplification.

A	B
Reagents	Vol. (μL)
Purified cDNA	1
25 μM TSO-PCR primer	0.2
2X KAPA FAST qPCR Readymix	5
H2O	3.8

Real-time PCR master mix

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

Prepare second strand synthesis mix:

A	B
Reagents	Vol. (μL)
10X Isothermal Amplification Buffer II	10
20% Ficoll PM-400	20
10 mM dNTPs	14
Bst 3.0 DNA Polymerase (NEB)	5
TSO-N3G2N4B primer (100 μM)	10
MgSO4 (100 mM)	6
H2O	35

Second strand synthesis master mix

After aspiration of TE-TW buffer, resuspend beads in 500 μL 0.1 M NaOH.

Incubate beads at room temperature for 5 min with rotation.

Add 500 μL 0.2M Tris-HCl (pH 7.5) to neutralize the solution.

Wash beads once with TE-TW and once with 10 mM Tris-HCl (pH 8.0).

Add 200 μL second strand synthesis mix to the beads. [Add Bst3 enzyme right before the reaction.]

Incubate beads at 15 min at 60 C with rotation.

Wash beads once with 1 mL TE-SDS, and twice with 1 mL TE-TW and twice with H2O.

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

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 ).

Elute the cDNA with 40 μL Low-EDTA TE.

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).

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.

Add 10 μL of Nextra TD buffer and 5 μL of Amplicon Tagmentation enzyme to each reaction. Mix by pipetting ~5 times.

Incubate at 55 C for 5 min.

Add 5 μL of Neutralization Buffer to each reaction. Mix by pipetting ~5 times. Spin down and incubate at room temperature for 5 min.

Add to each PCR tube in the following order:

A	B
Reagents	Vol. (μL)
Nextra PCR mix	15
2 μM P5-TSO hybrid primer	5
2 μM Nextera N70X oligo	5

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

Purify PCR product twice with 0.6X SPRI beads. Elute the cDNA in 12 μL H₂O.

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).

Dilute the library to 2 nM and pool the libraries according to the estimated cell numbers (100 cells/6000 beads).