TITLE: High-throughput Smart-seq3
AUTHORS: Kai-Hui Sun, Hsiu-Chun Chuang

[DESCRIPTION]
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.

[STEPS]
SECTION: Single Cell Collection via FACS
1. Prepare Cell Lysis Buffer Master Mix on the same day as single cell collection via FACS, and keep it on ice.

 

SECTION: Cell Lysis
5. Remove the 96-well plates from the -80 °C freezer and incubate them in a thermocycler at 72 °C for 10 min, followed by a hold at 4 °C.
SECTION: Reverse Transcription
6. Prepare Reverse Transcription (RT) Master Mix, and dispense 1 µL RT Master Mix into each well of the 384-well plates using Mantis.

 

SECTION: cDNA Amplification
9. Prepare PCR Master Mix, and keep it on ice.

 

SECTION: cDNA Purification
12. Program Integra VIAFLO to perform cDNA purification as follows:

Add 6 µL   (0.6X) to each well of the 384-well plate and mix by pipetting up and down.
Incubate 5 min at Room temperature.
Place on magnet until the solution clears.
Remove the supernatant.
Wash twice with 30 µL freshly prepared 80% ethanol.
Remove the ethanol, and air dry for 2 min.
Add 11 µL nuclease-free water and mix by pipetting up and down.
Incubate 5 min at Room temperature.
Place on magnet until the solution clears.
Transfer 10 µL purified sample to a new 384-well plate.
Store at -20 °C.
SECTION: cDNA Quantification
13. Use   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


SECTION: cDNA Normalization
14. Calculate and normalize each sample to 100 pg/ul using nuclease-free water as follows:

Prepare a new 384-well plate for cDNA normalization by dispensing calculated volumes of nuclease-free water (diluent) into each well using Mantis.
Transfer 1 µL purified cDNA into this new 384-well plate prepared above.
SECTION: Library Generation
15. Use   with our modified protocol to perform library generation.

SECTION: Library Generation
15.1. Prepare Tagmentation Master Mix, and dispense 3 µL into a new 384-well plate using Mantis.

 

SECTION: Library Generation
15.2. Add 1 µL normalized cDNA (100 pg/ul) to Tagmentation Master Mix, and mix by pipetting up and down using Integra VIAFLO.

SECTION: Library Generation
15.3. Seal the plate, perform a quick spin-down, and then incubate it in a thermocycler as follows:

55 °C for 5 min 
Hold at 4 °C
SECTION: Library Generation
15.4. Add 1 µL Neutralize tagment buffer (NT) using Mantis, mix and perform a quick spin-down, followed by incubation for 5 min at Room temperature.

SECTION: Library Generation
15.5. Dispense 3 µL Nextera PCR Master Mix (NPM) using Mantis, followed by adding 2 µL UDIs (1:1 dilution) and mixing using Integra VIAFLO.
SECTION: Library Generation
15.6. Seal the plate, perform a quick spin-down, and then incubate it in a thermocycler as follows:

72 °C for 3 min 
95 °C for 30 s 
14 cycles of:
95 °C for 10 s
55 °C for 30 s
72 °C for 30 s 
72 °C for 5 min 
Hold at 4 °C
SECTION: Pooled Library Purification
16. Pool libraries with different UDIs to perform library purification as follows:

Take 2 µL from each library with a different UDI from the plates in step 15, and pool them together for purification.
Add 0.6:1   to final volume of the pooled libraries, mix by pipetting up and down.
Incubate 5 min at Room temperature.
Place on magnet until the solution clears.
Remove the supernatant.
Wash twice with 1000 µL freshly prepared 80% ethanol.
Remove the ethanol, and air dry for 5 min.
Add 26 µL nuclease-free water and mix by pipetting up and down.
Incubate 5 min at Room temperature.
Place on magnet until the solution clears.
Transfer 25 µL purified sample to a new Eppendorf tube.
Store at -20 °C.

SECTION: Library Quality Control
17. Measure the concentration of pooled library using  , and determine the size using  .
SECTION: Sequencing
18. 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.
SECTION: Single Cell Collection via FACS
2. Dispense 4 µL Cell Lysis Buffer Master Mix into each well of the 96-well plates using Mantis. Keep the plates sealed and on ice prior to FACS.
SECTION: Single Cell Collection via FACS
3. 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.
SECTION: Single Cell Collection via FACS
4. 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 -80 °C.
SECTION: Reverse Transcription
7. 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.
SECTION: Reverse Transcription
8. Seal the plate, perform a quick spin-down, and then incubate it in a thermocycler as follows:

42 °C for 90 min 
10 cycles of:
50 °C for 2 min
42 °C for 2 min
85 °C for 5 min 
Hold at 4 °C
SECTION: cDNA Amplification
10. After completion of RT at step 8, perform a quick spin-down, and then dispense 6 µL PCR Master Mix into each well of the 384-well plate using Mantis.
SECTION: cDNA Amplification
11. Seal the plate, perform a quick spin-down, and then incubate it in a thermocycler as follows:

98 °C for 3 min 
18-25 cycles of:
98 °C for 20 s
65 °C for 30 s
72 °C for 4 min 
72 °C for 5 min 
Hold at 4 °C