Aug 12, 2024

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

DOI
https://dx.doi.org/10.17504/protocols.io.j8nlk8811l5r/v1
  • Qi Qiu1,
  • Fan Li1,
  • Dongming Liang1,
  • William Gao1,
  • Hao Wu1
  • 1Department of Genetics, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia PA 19104, USA
Fan Li
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlk8811l5r/v1
Protocol Citation: Qi Qiu, Fan Li, Dongming Liang, William Gao, Hao Wu 2024. scNT-seq2: single-cell metabolically labelled new RNA tagging sequencing for time-resolved analysis of gene expression in single cells. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8811l5r/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 05, 2024
Last Modified: August 12, 2024
Protocol  Integer ID: 101225
Keywords: new rna tagging, new rna, resolved rna, metabolic labeling, rna, rnas from the same cell, seq2 library preparation for illumina, seq2 library preparation, sequencing, analysis of gene expression, marking new transcript, gene expression, seq method for joint profiling, single cell, seq2 method, seq2, strand synthesis reaction
Abstract
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.
Materials
ABC
Chemicals, Peptides, and Recombinant ProteinsSourceIdentifier
DPBS, no calcium, no magnesiumInvitrogen14190136
Barcoded beadsChemGenesMACOSKO-2011-10(V+)
1M Tris-HCl, pH 8.0Invitrogen15568-025
1 M Tris-HCl, pH 7.5Invitrogen15567-027
0.5 M EDTA, pH 8.0Invitrogen15575-020
NxGen RNase InhibitorLucigen30281-2
Bovine Serum AlbuminSigma-AldrichA8806-5G
Ficoll PM-400GE Healthcare/Fisher Scientific45-001-745
SarkosylSigma-AldrichL7414-50mL
NaIO4Sigma-Aldrich7790-28-5
sodium acetateInvitrogenAM9740
Bst 3.0 DNA PolymeraseNEBM0374
Exonuclease INEBM0293L
TFEASigma-Aldrich269042-1G
SPRISelect beadsBeckman CoulterB23318
DTTFermentasR0862
QX200 Droplet Generation Oil for EvaGreenBio-Rad186-4006
Perfluoro-1-octanolSigma-Aldrich370533-25G
dNTPsClontech639125
Critical Commercial Assays
Maxima H Minus Reverse TranscriptaseThermoFisherEP0753
Nextera XT DNA sample preparation kit IlluminaFC-131-1096
KAPA HiFi hotstart readymixKAPA BiosystemsKK2602
Oligonucleotides
Template Switch OligoAAGCAGTGGTATCAACGCAGAGTGAATrGrGrG
TSO-PCR primerAAGCAGTGGTATCAACGCAGAGT
Custom Read 1 PrimerGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC
TSO-N3G2N4B primerAAGCAGTGGTATCAACGCAGAGTGA (N1:25252525)(N1)(N1)GG(N1)(N1)(N1)(N1)(N2: 00333433); N1 represents a mixture of A, C, G and T at a 25:25:25:25 ratio, N2 represents a mixture of A, C, G and T at a 0:33:34:33 ratio
P5-TSO hybrid primerAATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGT∗A∗C
Metabolic labeling
Prepare a 1 M stock solution of 4-thiouridine (4sU) by dissolving the powder in DMSO.
Note
Note: the stock is stable for a few months at -20 C. Protect the solution from light.

For metabolic labeling, the medium was replaced with fresh medium supplemented with nontoxic concentrations of 4sU (i.e. 100 or 200 μM).
Note
If the labeling time is longer than 4 hours, regular exchange of fresh 4sU-containing
media (i.e. every three hours) can enhance 4sU incorporation.

Prepare cell suspension
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).
Cell and beads co-encapsulation
Prepare lysis buffer:

ABC
ReagentsVol.(μL)Final Concentration
H2O400
20% Ficoll PM-4003006%
20% Sarkosyl100.2%
0.5 M EDTA4020 mM
1.0 M Tris-HCl, pH 7.5200200 mM
1.0 M DTT (add freshly)5050 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:

AB
Syringe ContentFlow Rate (μL/hr)
Oil15000
Cells4000
Beads4000

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).
Droplet breakage
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.
Chemical conversion of mRNAs on beads
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.

AB
ReagentsVolume (μL)
3 M sodium acetate (pH 5.2)8
0.5 M EDTA (pH 8.0)2
H2O214
TFEA13
Sub-total237
192 mM NaIO413
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.

AB
ReagentsFor 1mL (μL)
1 M Tris-HCl (pH7.5)10
1M DTT10
5M NaCl20
0.5M EDTA2
RNase Inhibitor20
DEPC-H2O958
Reducing Buffer

Reverse transcription
Prepare RT mix:

AB
ReagentsVol. (μL)
H2O80
Maxima 5X RT buffer40
20% Ficoll PM-40040
10 mM dNTPs20
100 μM Template Switch Oligo5
RNase Inhibitor5
Maxima H Minus Reverse Transcriptase10


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).]
Exonuclease I treatment
Prepare Exonuclease mix (makes 200 μL):

AB
ReagentsVol. (μL)
10X Exonuclease I buffer20
H2O170
Exonuclease I10

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).]
Run 1st round of TSO-PCR using 6,000-beads as templates
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:

AB
ReagentsVol. (μL)
KAPA HiFi HS Readymix25
100 μM TSO-PCR primer0.4
H2O24.6
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.

AB
ReagentsVol. (μL)
Purified cDNA1
25 μM TSO-PCR primer0.2
2X KAPA FAST qPCR Readymix5
H2O3.8
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
Second strand synthesis
Prepare second strand synthesis mix:
AB
ReagentsVol. (μL)
10X Isothermal Amplification Buffer II10
20% Ficoll PM-40020
10 mM dNTPs14
Bst 3.0 DNA Polymerase (NEB)5
TSO-N3G2N4B primer (100 μM)10
MgSO4 (100 mM)6
H2O35
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.
Large-scale TSO-PCR
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).
Tagmentation (Nextera® XT DNA Sample Preparation kit)
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:

AB
ReagentsVol. (μL)
Nextra PCR mix15
2 μM P5-TSO hybrid primer5
2 μM Nextera N70X oligo5

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).
Sequencing
Dilute the library to 2 nM and pool the libraries according to the estimated cell numbers (100 cells/6000 beads).
Protocol references
1. Hu, Peng, et al. "Dissecting cell-type composition and activity-dependent transcriptional state in mammalian brains by massively parallel single-nucleus RNA-seq." Molecular cell 68.5 (2017): 1006-1015.

2. Qiu, Qi, et al. "Massively parallel and time-resolved RNA sequencing in single cells with scNT-seq." Nature methods 17.10 (2020): 991-1001.