Oct 02, 2024

Public workspaceHigh-throughput Smart-seq3

DOI
dx.doi.org/10.17504/protocols.io.ewov196jolr2/v1
  • 1Gilead
Kai-Hui Sun
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.ewov196jolr2/v1
Protocol Citation: Kai-Hui Sun, Hsiu-Chun Chuang 2024. High-throughput Smart-seq3. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov196jolr2/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: September 25, 2024
Last Modified: October 02, 2024
Protocol Integer ID: 108293
Keywords: single-cell RNA sequencing, single-cell TCR sequencing, smart-seq3, high-throughput smart-seq3
Abstract
We built upon the Smart-seq3 protocol to develop the high-throughput Smart-seq3 (HT Smart-seq3) workflow, an automated workflow with a detailed and optimized protocol.
Materials
Protocol materials
ReagentNextera XT DNA Library Preparation KitIllumina, Inc.Catalog #FC-131-1096
Step 15
ReagentQubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
In 2 steps
ReagentBioAnalyzer High Sensitivity Chip Agilent TechnologiesCatalog #5067-4626
Step 17
Reagent Ampure XP beads Beckman CoulterCatalog #A63881
In 2 steps
Single Cell Collection via FACS
Single Cell Collection via FACS
Prepare Cell Lysis Buffer Master Mix on the same day as single cell collection via FACS, and keep it TemperatureOn ice .

ABCDE
Master mix componentCat # (Vendor)Stock conc.Reaction conc.Volume (ul) per well
Poly-ethylene glycol 800089510-250G-F (Sigma)50%5%0.4
Triton X-100T8787-50ML (Sigma)10%0.1%0.04
Recombinant RNAse inhibitor2313A (Takara Bio)40U/ul0.5U/ul0.05
Smart-seq3 Oligo-dT-30VNIntegrated Dna Technologies (IDT) 100uM0.5uM0.02
dNTPsR0182 (Thermo Fisher Scientific)25mM/each0.5mM/each0.08
Nuclease-free waterAM9930 (Invitrogen)3.41
Total4

Dispense Amount4 µL Cell Lysis Buffer Master Mix into each well of the 96-well plates using Mantis. Keep the plates sealed and TemperatureOn ice prior to FACS.
2m
Before loading the 96-well plate onto FACS, perform a quick spin-down and then sort single cells directly into wells containing Cell Lysis Buffer.
After completion of single cell collection, seal the 96-well plate, perform a quick spin-down, and place it on dry ice before transferring to storage at Temperature-80 °C .
Cell Lysis
Cell Lysis
10m
10m
Remove the 96-well plates from the Temperature-80 °C freezer and incubate them in a thermocycler at Temperature72 °C for Duration00:10:00 , followed by a hold at Temperature4 °C .
10m
Reverse Transcription
Reverse Transcription
2h 30m
2h 30m
Prepare Reverse Transcription (RT) Master Mix, and dispense Amount1 µL RT Master Mix into each well of the 384-well plates using Mantis.

ABCDE
Master mix componentCat # (Vendor)Stock conc.Reaction conc.Volume (ul) per well
Tris-HCI, pH 8.3T1083 (Teknova)1M25mM0.1
NaClAM9759 (Invitrogen)1M30mM0.12
MgCl2AM9530G (Invitrogen)100mM2.5mM0.1
GTPR1461 (Thermo Fisher)100mM1mM0.04
Dithiothreitol (DTT)707265ML (Thermo Fisher Scientific)100mM8mM0.32
Recombinant RNAse inhibitor2313A (Takara Bio)40U/ul0.5U/ul0.05
Smart-seq3 TSOIntegrated Dna Technologies (IDT) 100uM2uM0.08
Maxima H-minus RT enzymeEP0751 (Thermo Fisher)200U/ul2U/ul0.04
Nuclease-free waterAM9930 (Invitrogen)0.15
Total1

After completion of cell lysis at step 5, use Integra VIAFLO to transfer the lysates from four 96-well plates into one 384-well plate pre-filled with RT Master Mix at step 6, and then mix well.
1m
Seal the plate, perform a quick spin-down, and then incubate it in a thermocycler as follows:

Temperature42 °C for Duration01:30:00
10 cycles of:
  • Temperature50 °C for Duration00:02:00
  • Temperature42 °C for Duration00:02:00
Temperature85 °C for Duration00:05:00
Hold at Temperature4 °C
2h 15m
cDNA Amplification
cDNA Amplification
2h 30m
2h 30m
Prepare PCR Master Mix, and keep it TemperatureOn ice .

ABCDE
Master mix componentVendorStock conc.Reaction conc.Volume (ul)
Kapa HiFi HotStart bufferKK2502 (Roche)5X1X2
DNA polymeraseKK2502 (Roche)1U/ul0.02U/ul0.2
dNTPsR0182 (Thermo Fisher Scientific)25mM/each0.3mM/each0.12
MgCl2AM9530G (Invitrogen)100mM0.5mM0.05
Smart-seq3 forward primerIntegrated Dna Technologies (IDT) 100uM0.5uM0.05
Smart-seq3 reverse primerIntegrated Dna Technologies (IDT) 100uM0.1uM0.01
Nuclease-free waterAM9930 (Invitrogen)3.57
Total6

After completion of RT at step 8, perform a quick spin-down, and then dispense Amount6 µL PCR Master Mix into each well of the 384-well plate using Mantis.
2m
Seal the plate, perform a quick spin-down, and then incubate it in a thermocycler as follows:

Temperature98 °C for Duration00:03:00
18-25 cycles of:
  • Temperature98 °C for Duration00:00:20
  • Temperature65 °C for Duration00:00:30
  • Temperature72 °C for Duration00:04:00
Temperature72 °C for Duration00:05:00
Hold at Temperature4 °C
2h 15m
cDNA Purification
cDNA Purification
30m
30m
Program Integra VIAFLO to perform cDNA purification as follows:

  1. Add Amount6 µL Reagent Ampure XP beads Beckman CoulterCatalog #A63881 (0.6X) to each well of the 384-well plate and mix by pipetting up and down.
  2. Incubate Duration00:05:00 at TemperatureRoom temperature .
  3. Place on magnet until the solution clears.
  4. Remove the supernatant.
  5. Wash twice with Amount30 µL freshly prepared 80% ethanol.
  6. Remove the ethanol, and air dry for Duration00:02:00 .
  7. Add Amount11 µL nuclease-free water and mix by pipetting up and down.
  8. Incubate Duration00:05:00 at TemperatureRoom temperature .
  9. Place on magnet until the solution clears.
  10. Transfer Amount10 µL purified sample to a new 384-well plate.
  11. Store at Temperature-20 °C .
30m
cDNA Quantification
cDNA Quantification
10m
10m
Use ReagentQubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854 with our modified protocol to perform cDNA quantification as followed:

1. Prepare the Qubit‱ Working Solution: Dilute the Qubit dsDNA HS Reagent at a ratio of 1:200 in Qubit‱ dsDNA HS Buffer

2. Prepare Standards: Create a series of standards at concentrations of 0, 1.25, 2.5, and 5 ng/µL by performing serial dilutions of Qubit‱ 1X dsDNA HS Standard #2, which has a concentration of 10 ng/µL. This will allow for accurate quantification during the assay.

3. Dispense Working Solution: Using an Integra VIAFLO, dispense 34 µL of the prepared Qubit‱ working solution into each well of the black, flat-bottom 384-well plates (Corning, Catalog#3820)

4. Add Standards: In a separate plate, add 1 µL of each prepared standard to the corresponding wells. This will enable the creation of a standard curve for quantifying the cDNA concentration.

5. Add cDNA Samples: Add 1 µL of cDNA sample to each well of the plate containing the Qubit‱ working
solution. Ensure that samples are added to the correct wells to maintain accurate results.

6. Measure Fluorescence: Use a SpectraMax microplate reader to measure the fluorescence of the samples. Set the excitation wavelength to 485 nm and the emission wavelength to 525 nm.
Record the fluorescence intensity for each well.

7. Calculate cDNA Concentration: Analyze the fluorescence data to calculate the cDNA concentration in the samples using the standard curve generated from the known standards


10m
cDNA Normalization
cDNA Normalization
10m
10m
Calculate and normalize each sample to Concentration100 pg/ul using nuclease-free water as follows:

  1. Prepare a new 384-well plate for cDNA normalization by dispensing calculated volumes of nuclease-free water (diluent) into each well using Mantis.
  2. Transfer Amount1 µL purified cDNA into this new 384-well plate prepared above.
10m
Library Generation
Library Generation
5m
5m
Use ReagentNextera XT DNA Library Preparation KitIllumina, Inc.Catalog #FC-131-1096 with our modified protocol to perform library generation.

Prepare Tagmentation Master Mix, and dispense Amount3 µL into a new 384-well plate using Mantis.

AB
Mater mix component (Kit)Volume (ul)
Tagment DNA buffer (TD)2
Amplicon tagment mix (ATM)1
Total3

2m
Add Amount1 µL normalized cDNA (Concentration100 pg/ul ) to Tagmentation Master Mix, and mix by pipetting up and down using Integra VIAFLO.

1m
Seal the plate, perform a quick spin-down, and then incubate it in a thermocycler as follows:

Temperature55 °C for Duration00:05:00
Hold at Temperature4 °C
5m
Add Amount1 µL Neutralize tagment buffer (NT) using Mantis, mix and perform a quick spin-down, followed by incubation for Duration00:05:00 at TemperatureRoom temperature .

5m
Dispense Amount3 µL Nextera PCR Master Mix (NPM) using Mantis, followed by adding Amount2 µL UDIs (1:1 dilution) and mixing using Integra VIAFLO.
3m
Seal the plate, perform a quick spin-down, and then incubate it in a thermocycler as follows:

Temperature72 °C for Duration00:03:00
Temperature95 °C for Duration00:00:30
14 cycles of:
  • Temperature95 °C for Duration00:00:10
  • Temperature55 °C for Duration00:00:30
  • Temperature72 °C for Duration00:00:30
Temperature72 °C for Duration00:05:00
Hold at Temperature4 °C
9m 40s
Pooled Library Purification
Pooled Library Purification
15m
15m
Pool libraries with different UDIs to perform library purification as follows:

  1. Take Amount2 µL from each library with a different UDI from the plates in step 15, and pool them together for purification.
  2. Add 0.6:1 Reagent Ampure XP beads Beckman CoulterCatalog #A63881 to final volume of the pooled libraries, mix by pipetting up and down.
  3. Incubate Duration00:05:00 at TemperatureRoom temperature .
  4. Place on magnet until the solution clears.
  5. Remove the supernatant.
  6. Wash twice with Amount1000 µL freshly prepared 80% ethanol.
  7. Remove the ethanol, and air dry for Duration00:05:00 .
  8. Add Amount26 µL nuclease-free water and mix by pipetting up and down.
  9. Incubate Duration00:05:00 at TemperatureRoom temperature .
  10. Place on magnet until the solution clears.
  11. Transfer Amount25 µL purified sample to a new Eppendorf tube.
  12. Store at Temperature-20 °C .

15m
Library Quality Control
Library Quality Control
Measure the concentration of pooled library using ReagentQubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854 , and determine the size using ReagentBioAnalyzer High Sensitivity Chip Agilent TechnologiesCatalog #5067-4626 .
Sequencing
Sequencing
Sequence the pooled library on an Illumina sequencer (Illumina, San Diego, CA, USA) using paired-end (PE) 150 bp reads, with a target of 1-2 million reads per sample.
Protocol references
1. Hagemann-Jensen M, Ziegenhain C, Chen P, Ramskold D, Hendriks GJ, Larsson AJM, Faridani OR, Sandberg R: Single-cell RNA counting at allele and isoform resolution using Smart-seq3. Nat Biotechnol 2020, 38(6):708-714.
2. Michael Hagemann-Jensen CZ, Ping Chen, Daniel Ramsköld, Gert-Jan Hendriks, Anton J.M Larsson, Omid R. Faridani, Rickard Sandberg: Smart-seq3 Protocol V.3. In. protocols.io; 2020.