TITLE: FLASH-seq protocol 
AUTHORS: Simone   Picelli, Vincent  Hahaut

[DESCRIPTION]
The single-cell RNA-sequencing (scRNA-seq) field has evolved tremendously since the first paper was published back in 2009. While the first methods analysed just a handful of cells, the throughput and performance rapidly increased over a very short timespan. However, it was not until the introduction of emulsion droplets methods, that the robust and reproducible analysis of thousands of cells became feasible. Despite generating data at a speed and a cost per cell that remains unmatched by full-length protocols like Smart-seq, scRNA-seq in droplets still comes with the drawback of addressing only the terminal portion of the transcripts, thus lacking the required sensitivity for comprehensively analyzing the transcriptome of individual cells. Building upon the existing Smart-seq2/3 workflows, we developed FLASH-seq (FS), a new full-length scRNA-seq method capable of detecting a significantly higher number of genes than both previous versions, requiring limited hands-on time and with a great potential for customization.

[BEFORE_START]
The protocol should be carried out in a clean environment, ideally on a dedicated PCR workstation or on a separate bench used only for this purpose. Before starting, clean the bench and wipe any piece of equipment with RNAseZAP or 0.5% sodium hypochlorite. Rinse with nuclease-free water to avoid corrosion of delicate equipment.

Work quickly and preferably on ice.

Reagent mixes should be prepared shortly before use.

Mix thoroughly each mix before dispensing. For higher accuracy use liquid handling robots and/or nanodispensers whenever possible. In FLASH-Seq we use the I.DOT (​​Dispendix) for all the dispensing steps and the Fluent 780 liquid handling robot (Tecan) for sample cleanup, reagent transfers and pooling.

The protocol described below is meant to be carried out in 384-well plates. When using 96-well plates, we recommend using 5 times larger volume to guarantee successful cell sorting and prevent evaporation issues.

Always use LoBind plates and tubes (especially for long-term storage) to prevent the cDNA/DNA from sticking to plastic.

[STEPS]
SECTION: Prepare lysis mix
1. 
 Prepare the following lysis mix:
    ReagentReaction concentrationVolume (µl)384-well plateTriton-X100 (10% v/v)0.2%0.0208.448dNTP mix (25 mM each)6 mM0.240101.376SMART dT30VN (100 µM)1.8 mM0.0187.603RNAse inhibitor (40 U/µL)1.2 U/µL0.03012.672DTT (100 mM)1.2 mM0.0125.069FS TSO (100 µM)9.2 µM0.09238.861dCTP (100 µM)9 mM0.09038.016Betaine (5 M)1 M0.20084.480Nuclease-free water -0.298125.875Total volume (µL) 1.000422.400

 Add 1 µL  lysis mix to each well of a 384-well plate

Seal the plate with a PCR seal and quickly spin it down to collect the lysis mix to the bottom.
 
Proceed immediately to the next step or store the plate at -20 °C  long-term. Plates that are going to be used on the same day can be stored in the fridge or kept on ice.
 

SECTION: Sample collection
2. Sort single cells into 384-well plates containing  1 µL lysis mix.

Seal the plate with an aluminium seal. If processing multiple plates at once, keep each plate on dry ice until ready to transfer them all at -80 °C  for long-term storage. Plates containing single cells should ideally be processed within 6 months.
SECTION: Cell lysis
3. Remove the plates from the -80 °C  freezer and check that the aluminium seal is still intact. If damaged or not sticking to the plate anymore, wait a few minutes for the plate to partially thaw, remove the damaged foil and replace it with a new one.

Place the plate in a thermocycler with a heated lid and incubate for 3 min  at 72 °C , followed by a 4 °C  hold step.

Spin down any condensation droplets that may have formed during the incubation and return the plate to a cool rack. Proceed quickly to the next step. If not ready with the RT-PCR mix, keep the plate on the cool rack at all times.
SECTION: RT-PCR reaction
4. While the plate is in the thermocycler, prepare the following RT-PCR mix:
    ReagentReaction concentrationVolume (µL)384-well plateDTT (0.1 M)4.8 mM0.238100.531MgCl2 (1 M)9.2 mM0.04619.430Betaine (5 M)800 mM0.800337.920RNAse inhibitor (40 U/µL)0.8 U/µL0.09640.550SuperScript IV (200 U/µlL2.00 U/µL0.05021.120KAPA HiFi HotStart Ready Mix (2x)1x2.5001056.000Nuclease-free water-0.270114.048Total volume (µL)4.0001689.600

 Add 4 µL  RT-PCR mix into each well of the 384-well plate.

Seal the plate with a PCR seal, gently vortex and spin down to collect the liquid at the bottom.

Place it in a thermocycler with heated lid and start the following RT-PCR program:

     StepTemperatureTimeCyclesRT50ºC60 min1xPCRInitial denaturation98ºC3 min1xDenaturation98ºC20 sec18-21x*Annealing67ºC20 secElongation72ºC6 min15ºCHold

 

 
 
SECTION: Magnetic beads working solution preparation
5. You can either use AMPure XP beads, SPRI beads or prepare your own solution of SeraMag beads containing 18% w/v PEG to reduce costs. A detailed protocol for making your own magnetic bead solution is described in:
 

 Below is a short description of how to prepare 50 ml of working solution:
   ReagentAmount to addFinal concentrationSodium chloride2.92 gr1 MTris-HCl, pH=8.0 (1 M)500 µL10 mMEDTA (500 mM)100 µL1 mMPEG (MW=8000)9.5 gr18% w/vNuclease-free solutionto a final volume of 50 mL-

Add all components to a 50-ml Falcon tube but do not add the total amount of nuclease-free water yet.

Solubilise the PEG by stirring and heating the solution at 37 °C .

While the PEG is dissolving, prepare the Sera-Mag SpeedBeads™. Vortex thoroughly to ensure complete resuspension and then withdraw 1 mL  Sera-Mag SpeedBeads™ stock solution. Transfer it into a new 1.5-ml tube. 

Pellet the beads by placing the tube on a magnetic stand. Wait until the solution clears and then discard the supernatant. 

Add 1 mL  10 mM Tris–HCl pH 8.0 + 1 mM EDTA (TE buffer) and resuspend the beads off the magnet.

Pellet the beads again, wait until the solution is clear, discard the supernatant and resuspend off the magnet with 0.9 mL  TE buffer. 

Once the PEG solution is clear, add the resuspended beads prepared in the previous step. 

Add 50 µL  Tween-20 (10% v/v) and 250 µL  sodium azide (NaN3, 10% w/v) and adjust the volume to 50 mL  with nuclease-free water. 

Store at 4 °C . Do not freeze. 
 

 

 

SECTION: cDNA purification
6. Remove the Sera-Mag SpeedBeads™ working solution (or AMPure XP beads or SPRI beads when using a commercial solution) from the 4 °C  storage and equilibrate it at room temperature for 15 min .

 

Add a 0.8 x volume ratio Sera-Mag SpeedBeads™ working solution to each well. Mix thoroughly by pipetting or vortexing.

Incubate the plate off the magnetic stand for 5 min  at Room temperature .

Place the plate on the magnetic stand and leave it for 5 min  or until the solution appears clear.

Remove the supernatant without disturbing the beads. 

Remove the plate from the magnetic stand, add 15 µL  nuclease-free water and mix well by pipetting or vortexing to resuspend the beads. Do not let the bead pellet to dry completely, as it can decrease the final cDNA yield!

Incubate 2 min  off the magnetic stand.

Place the plate back on the magnetic stand and incubate for 2 min  or until the solution appears clear.

Remove 14 µL  of the supernatant and transfer it to a new plate.

 

SECTION: Quality control check (highly recommended!)
7. Check the cDNA quality on Agilent Bioanalyzer High Sensitivity DNA chip. A good sample is characterised by a low proportion of fragments <400 bp, absence of residual primers (ca. 100 bp) and an average cDNA size of 1.8–2.2 Kb.


 


SECTION: cDNA quantification (optional but recommended)
8. Allow the Quant-iT PicoGreen reagent to warm to Room temperature  before opening the vial. PicoGreen is light sensitive; while thawing, wrap in aluminium foil. 

Prepare a 1 x working solution TE using 20 x TE (supplied) and nuclease-free water. 

Prepare a 1:400 dilution PicoGreen solution and always use a plastic vessel.

Prepare the standard curve using Lambda DNA standard (supplied at a concentration of 100 ng/μl , with thePicoGreen kit) and 1 x TE in 8 tubes, as below. The stock tubes can be used multiple times, keep any leftover in the fridge at 4 °C  between experiments.

Vortex well and spin down the DNA standards before every use. Not vortexing thoroughly the standards is going to negatively affect the standard curve and your readings! Serial dilutions should be prepared as shown in the table below.
 
    Tube no.ContentsConcentrationFinal volume190 μL TE + 10 μL Lambda DNA stock10 ng/μL100 μL250 μL from Tube 1 + 50 μL TE5 ng/μL100 μL350 μL from Tube 2 + 50 μL TE2.5 ng/μL100 μL450 μL from Tube 3 + 50 μL TE1.25 ng/μL100 μL550 μL from Tube 4 + 50 μL TE0.625 ng/μL100 μL650 μL from Tube 5 + 50 μL TE0.3125 ng/μL100 μL750 μL from Tube 6 + 50 μL TE0.15625 ng/μL100 μL8TE onlyblank-

Prepare the PicoGreen solution by pipetting 0.5 µL  PicoGreen dye + 99.5 µL  1 X TE for each sample. Vortex to mix.

Pipette 1 µL  each of the 7 standards + 1 Blank into a black, flat-bottom Nunc™ F96 MicroWell™ plate. Place the standards on one column.

Pipet 1 µL  of your samples into the center of each well of the Nunc™ F96 MicroWell™ polystyrene plate.

Add 99 µL  PicoGreen + TE mix into every well. There is no need to mix.

Cover the plate with the provided plastic (transparent) lid to prevent possible contaminations.

Allow 2 min  for the dye to bind the DNA. Protect from light but keep at room temperature. For optimal results, the plate should be read within the next hour.

Use a plate reader to measure fluorescence (excitation: 485 nm; emission: 530 nm; read from top; endpoint reading). 
SECTION: Plate normalisation
9. Prepare a normalisation plate by adding 1 µL  purified cDNA and nuclease-free water to a final concentration of 150 pg/μl .


SECTION: Tagmentation and enrichment PCR
10. This step can be carried out either by using the commercially available Nextera XT kit or a in-house Tn5 transposase, as described below. Indexing primers can be purchased from Illumina (Nextera XT index kit v2) or ordered from your local oligo manufacturer. In the "Materials" section we have added additional sequences for higher multiplexing.
SECTION: Tagmentation and enrichment PCR
10.1. Tagmentation with the Nextera XT kit
 

 Prepare the tagmentation mix as described below:
  ReagentVolume (µL)ATM (Amplification Tagment Mix)0.250TD (Tagmentation DNA Buffer)2.000Nuclease-free water0.500Total volume (µL)3.000

 Dispense 3 µL  tagmentation mix in a new 96-well or 384-well plate.

Add 1 µL  normalized cDNA (150 pg/μl ) to each well containing the tagmentation mix.

Seal the plate, vortex, spin down, and carry out the tagmentation reaction: 55 °C  for 8 min , 4 °C  hold. Upon completion proceed immediately to the next step.

Add 1 µL  NT buffer to each well. Seal the plate, vortex, spin down and incubate 5 min  at room temperature. Do not put the plate back on ice.

Add 2 µL  N7xx + S5xx index adaptors (5 micromolar (µM)  each).

Add 3 µL  NPM solution to each well.

Seal the plate, vortex, spin down, and place it in a thermocycler and carry out the enrichment PCR reaction. Adjust the number of PCR cycles according to the number of processed cells.
     StepTemperatureTimeCyclesGap filling72ºC3 min1xEnrichment PCRInitial denaturation95ºC30 sec1xDenaturation95ºC10 sec12-14xAnnealing55ºC30 secElongation72ºC30 sec15ºChold


 

SECTION: Tagmentation and enrichment PCR
10.2. Tagmentation with in-house Tn5 transposase
 

Prepare the tagmentation mix as described below:
   ReagentVolume (µl)Final concentrationTAPS-Mg buffer, pH=7.3 (5x)0.80010 mM TAPS, 5 mM MgCl2Dimethylformamide (DMF) (100%)0.80020%Tn5 transposase (2 µM working dil.)0.12562.5 nmolNuclease-free water2.275Total volume (µl)3.000

 

 Dispense 3 µL  tagmentation mix in a new 384-well plate.

Add 1 µL  normalized cDNA (150 pg/μl ) to each well containing the tagmentation mix.

Seal the plate, vortex, spin down, and carry out the tagmentation reaction: 55 °C  for 8 min , 4 °C  hold. Upon completion proceed immediately to the next step.

Add 1 µL  0.2% SDS to each well. Seal the plate, vortex, spin down and incubate 5 min at room temperature. Do not put the plate back on ice.

Add 2 µL  N7xx + S5xx index adaptors (5 micromolar (µM)  each).

Add 3 µL  enrichment PCR mix to each well:
   ReagentVolume (µl)Final concentrationKAPA HiFi enzyme (1 U/μL)0.2000.02 U/μLKAPA HiFi Buffer (5x)2.0001xdNTPs (10 mM)0.300300 nMNuclease-free water0.500Total volume (µl)3.000

Seal the plate, vortex, spin down, and place it in a thermocycler and carry out the enrichment PCR reaction. Adjust the number of PCR cycles according to the number of processed cells.
     StepTemperatureTimeCyclesGap filling72ºC3 min1xEnrichment PCRInitial denaturation98ºC30 sec1xDenaturation98ºC10 sec12-14xAnnealing55ºC30 secElongation72ºC30 sec15ºChold


 
SECTION: Library cleanup and quantification
11. Take an aliquot from each sample for the final library cleanup (i.e. 5 µl). and transfer it to a 1.5-ml Eppendorf tube. The rest of the library can be stored long-term at -20 °C .

Remove the Sera-Mag SpeedBeads™ working solution from the 4 °C  storage and equilibrate it at room temperature for 15 min .

Add Sera-Mag SpeedBeads™ working solution to a final ratio of 0.8 x and mix well to homogenisation.

Incubate the tube off the magnetic stand for 5 min  at Room temperature .

Place the tube on the magnetic stand and leave it for 5 min  or until the solution appears clear.

Remove the supernatant without disturbing the beads.

Recommended: wash the pellet with 1 mL  80% v/v ethanol. Incubate 30 s  without removing the tube from the magnetic stand.

Remove any trace of ethanol and let the bead pellet dry for 2 min  or until small cracks appear. Do not cap the tube or remove it from the magnetic stand during this time. Do not completely air-dry the beads.

Remove the tube from the magnetic stand, add 50 µL  nuclease-free water and mix well by pipetting or vortexing to resuspend the beads.

Incubate 2 min  off the magnetic stand.

Place the tube back on the magnetic stand and incubate for 2 min or until the solution appears clear.

Remove 49 µL  of the supernatant and transfer it to a new 1.5-ml LoBind tube. Store the cDNA at -20 °C long-term or until ready for sequencing.

Use Qubit fluorometer to quantify the library. Library yield can vary depending on the number of cells being pooled.

Check the final library size on the Agilent Bioanalyzer.

Use the average size indicated on the Bioanalyzer and the concentration reported after Qubit measurement to determine the exact molarity required for sequencing.

 


 

SECTION: Pooling and sequencing
12. The purified library can be sequenced on any Illumina sequencer. Follow the specifications reported for each instrument. Single-End 75 bp is generally sufficient but longer read modes or Paired-End sequencing can be an option, depending on the question at hand.
SECTION: Data processing
13. These instructions briefly describe the data processing of the sequencing results. The final pipeline will likely have to be adapted to the question at hand. The following lines assume that all the programs and their dependencies are installed on your machine and that the data are single-end reads (75 bp). Some values, such as the number of threads and RAM usage may have to be adapted to your machine settings. 

It should be noted that there are many other ways to analyse full-length single-cell RNA-sequencing data. Pseudo-alignment tools (e.g., Salmon or Kallisto) or automatic pipelines (zUMIs) could be used as well.

Requirements (tested version):

bcl2fastq (v2.20)
STAR (v2.7.3)
FeatureCounts (v1.6.5)
BBMAP (v38.86)
samtools (v1.9)
IGV

SECTION: Data processing
13.1. Sample demultiplexing

Sequencing results will be delivered as demultiplexed FASTQ or raw bcl2 files. To convert bcl2 files to FASTQ, bcl2fastq program (Illumina) can be used. 
 

 
When sequencing on a NextSeq550 instrument, the sample sheet should contain the following information in a csv file:

  
Illumina Experiment Manager can be used to assist you in creating the sample sheet. 

We recommend exploring the barcode combinations left in the undetermined reads looking to confirm that all the cells have been properly demultiplexed.


 
as well as the read distribution between samples:

 

SECTION: Data processing
13.2. Index the genome

The reference genome needs to be indexed prior to any mapping. The FASTA and GTF references can be obtained from ENSEMBL, Gencode, UCSC, ... 

 

 

 

SECTION: Data processing
13.3. FASTQ trimming (optional)

If you observe sequencing primer left-overs the FASTQ files can be trimmed using BBDUK or Trimmomatic.

 

 

SECTION: Data processing
13.4. Mapping

The FASTQ file can then be mapped onto the reference genome. Example for one sample, use a loop or parallelise this task to process all the cells:

 

 

 

SECTION: Data processing
13.5. Data visualization (optional)

Once the reads have been mapped we highly recommend using the Integrated Genome Viewer (IGV) to visualise the mapping results and ensure that the results make sense. As a quick check-up visualise a few housekeeping genes (i.e., ACTB, GAPDH, …) and cell specific markers to look for reads mapping to exon, intron, exon-intron junctions. Look for abnormalities such as read piles falling in intergenic or centromeric regions.
No single-cell RNA sequencing protocol is perfect and non-specific priming, genomic DNA contaminations, … can happen but should represent rare events.
Recurrent soft-clipping could also indicate the presence of sequencing adaptor left-overs that could affect the mapping rate. 
SECTION: Data processing
13.6. Count matrix

Finally, t​he number of reads associated with each gene can be obtained as follows:

 

SECTION: Data processing
13.7. Post-processing

The post-processing steps will vary depending on the question at hand. The online book “Orchestrating Single-Cell Analysis with Bioconductor” (https://bioconductor.org/books/release/OSCA/) is a gold mine of information that can be used to help you design your own pipeline. Alternatively, Seurat (R, https://satijalab.org/seurat/) or scanpy (python, https://scanpy.readthedocs.io/en/stable/) provide tools compatible with FLASH-seq data. Given their similarities, we currently recommend using Smart-seq2 guidelines when processing FLASH-seq data.