TITLE: Bulk FLASH-seq
AUTHORS: Rebecca A Siwicki, Vincent Hahaut, Simone Picelli

[DESCRIPTION]
Bulk RNA sequencing has revolutionized the study of transcriptomes, enabling the analysis of gene expression in complex tissues and heterogenous cell populations. While single cell RNA sequencing (scRNA-seq) has gained popularity due to its ability to profile individual cells, it comes with limitations such as high costs and reduced sensitivity for detecting low-abundance transcripts. 

Here, we present bulk FLASH-seq (FS), a full-length RNA-seq method based on the single cell FLASH-seq workflow (Hahaut et al, 2022, https://www.nature.com/articles/s41587-022-01312-3),  updated for bulk RNA analysis. FS bulk generates high quality data while requiring minimal hands-on time and offering a greater degree of customization. As a homebrew protocol, it is inexpensive compared to commercial kits allowing you to invest in greater sequencing depths or in a higher number of sequenced samples.  

Our protocol enables comprehensive transcriptome analysis of bulk RNA samples, providing an alternative approach to scRNA-seq for gene expression when single-cell RNA-sequencing is not required.

[BEFORE_START]
The protocol should be carried out in a clean environment, ideally on a dedicated PRE-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. For 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.

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

[GUIDELINES]
Bulk FS has been optimised for 1 ng RNA input. Any other input in the range 10 pg-1 ng will not require any change to the protocol we describe here. Any input <5 ng RNA might also be fine with the 5ul reaction volume. However, inputs >10 ng will almost certainly require a reagent titration, in particular dNTPs, oligodT, TSO and perhaps, RT. The reaction can also be scaled to 96-well plates but reaction volume will need to be increased from 5uL. The user should be aware of this and perform the necessary titrations. Bulk FS is, similarly to scFS, a polyA-only, non-stranded protocol and therefore unsuitable when preserving strand information is crucial.

[STEPS]
SECTION: Prepare Lysis Mix
1. Prepare the following Lysis Mix:
 
ABC  Reagent    Final Concentration    Volume 96 rxn    Triton-X100 (10% v/v)    0.02    2.208    dNTP mix (25 mM each)    0.24    26.496    SMART dT30VN (100uM)    0.018    1.9872    RNase Inhibitor (40 U/ul)    0.03    3.312    DTT (100 mM)    0.012    1.3248    dCTP    0.09    9.936    Betaine    0.2    22.08    Nuclease- free water    0.19    20.976    Total volume (μL)    0.8    88.32  

Add 0.68 µL lysis mix to each well of a 384-well plate.

Seal the plate with a PCR seal and quickly spin down to collect lysis mix at the bottom of the wells.

Process immediately to the next step or store plate long term at -20 °C. Plates that will be used the same day can be stored in the fridge 4 °C or on ice.

Safe stopping point. Plates containing lysis buffer can be stored for 6+ months at -20 °C
SECTION: Sample Normalisation &amp; Addition
2. Quantify input RNA using a spectrophotometry and fluorometry assay (ex: NanoDrop™ or Qubit™ RNA High Sensitivity).

Normalise input RNA to 2 ng/μL with Nuclease-free water. 

Add0.5 µL of normalised RNA into corresponding 384-well plate containing lysis mix.

 

Seal the plate with an aluminium seal, quickly spin down. If processing more than one plate at once, keep each plate on dry ice until ready to transfer all to -80 °Cfor long term storage. Plates containing RNA should ideally be processed within 6 months.
SECTION: Cell Lysis
3. Remove plates from -80 °Cand check that aluminum seal is still intact. If damaged or not sticking to the plate, wait a few minutes for the plate to partially thaw, remove damaged foil and replace with new one.

Place plate in a thermocycler with a heated lid and incubate for 3 min minutes at72 °C, followed by a 4 °C hold step.

 

Spin down any condensation droplets (30 s,   )  that may have formed during incubation and return the plate to a cold block. Process quickly to the next step. If not ready with RT-PCR mix, keep the plate on the cold block at all times.

SECTION: RT-PCR Reaction
4. Prepare RT-PCR mix:
 
     Reagent      Volume (μL)      Volume 96 rxn        DTT      0.238      26.2752        MgCl2 (1M)      0.046      5.0784        Betaine (5M)      0.8      88.32        Nuclease-free water      0.088      9.7152        Takara RNAse inhibitor (40 U/µl)      0.096      10.5984        Superscript IV (200U/ul)      0.05      5.52        KAPA HiFi HotStart Ready Mix (2X)      2.5      276        FS TSO      0.092      10.1568        Total volume (μL)      4      260.8   
Add 3.822 µL RT-PCR mix into each well of the 384-well plate.

Seal the plate with a PCR seal, gently vortex and spin down (30 s,  ) to collect the liquid at the bottom of the well.

Place plate in a thermocycler with a heated lid and start the following RT-PCR program:
 
     Step      Temperature      Time      Cycles        RT      50   C      60   min      1        PCR-   initial denaturation      98   C      3   min      1        PCR   - denaturation      98   C      20   sec      14X - 16 (?)        PCR   - annlealing      67   C      20   sec        PCR   - elongation      72   C      6   min               72   C      5   min      1X               4   C      hold          

 Safe Stopping Point. Amplified cDNA before purification can be stored for several months at -20 °C .
SECTION: Magnetic bead 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:
 
        <b>Reagent<b>  <b>Final   concentration<b>  <b>Amount   to add<b>    Sodium   chloride  1 M  2.92 gr    Tris-HCl, pH =   8.0 (1 M)  10 mM  500 µl    EDTA (500 mM)  1 mM  100 µl    PEG (MW=8000)  18% w/v  9.5 gr    Nuclease-free   water  -  to a final volume of   50 ml  
 
Combine the sodium chloride, Tris-HCl, EDTA and PEG in a a 50-ml Falcon tube. Add 25 mL water.

Solubilise the PEG by stirring and heating the solution at 37 °C. If needed, progressively add more water.

While the PEG is dissolving, prepare the Sera-Mag SpeedBeads™. Vortex thoroughly to ensure complete resuspension. Withdraw1 mL  Sera-Mag SpeedBeads™ stock solution and 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 TE buffer (10 mM Tris–HCl pH 8.0 + 1 mM EDTA) 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 (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 0.8X 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. Do not let the bead pellet dry completely as it can decrease the final cDNA yield.

Remove the plate from the magnetic stand, add 15 µL nuclease free water and mix well by pipetting or vortexing to resuspend the beads. 

Incubate for 2 min off the magnetic stand.

Place the plate back on the magnetic stand and incubate for2 minor until the solution appears clear.

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

Safe stopping point. Amplified and clean cDNA can be stored for several months at -20 C. We recommend storing in LoBind plates to avoid material loss during long-term storage.
SECTION: Quality control check (highly recommended)
7. Check the cDNA quality on the Agilent Bioanalyzer High Sensitivity DNA chip. A good sample is characterised by a low proportion of fragments <400 bp, absence of residual primers (~100bp) and an average cDNA size of 1.8-2.2 Kb.

 

SECTION: cDNA quantification (optional but recommended)
8. Allow the Quant-iT™ PicoGreen™ dsDNA Assay reagents to come to room temperature before opening the vial. PicoGreen™ dye is light sensitive, it should be thawed in dark drawer or wrapped in foil.

This step can be performed in either 384-well or 96-well plates. Volume of PicoGreen + TE solution should be adjusted depending on plate type.

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

Prepare the standard curve using Lambda DNA standard (supplied at concentration of 100 ng/μl, with the PicoGreen™ dsDNA Assay kit) and 1X 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 standard before every use. Not vortexing the standards thoroughly will negatively affect the standard curve and concentration readings. Serial dilutions should be prepared as shown in the table below and stored at 4 °C :
 
     Tube      Contents      Concentration      Final   Volume        1      90   μl TE + 10 μl Lambda DNA stock      10   ng/μL      100   μL        2      50   μl from Tube 1 + 50 μl TE      5   ng/μL      100   μL        3      50   μl from Tube 2 + 50 μl TE      2.5   ng/μL      100   μL        4      50   μl from Tube 3 + 50 μl TE      1.25   ng/μL      100   μL        5      50   μl from Tube 4 + 50 μl TE      0.625   ng/μL      100   μL        6      50   μl from Tube 5 + 50 μl TE      0.3125   ng/μL      100   μL        7      50   μl from Tube 6 + 50 μl TE      0.15625   ng/μL      100   μL        8      TE   only      blank          

For 384-well plates, prepare a 1:400 solution of PicoGreen™ dsDNA Assay in 1X TE buffer (39 µL  per sample). Vortex to mix.

 

Pipette 1 µL each of the 7 standards + 1 Blank into a black, flat-bottom 384 MicroWell™ plate. Place the standards on one column.

Pipette 1 µL of your samples into the center of each well of the Nunc™ F384 MicroWell™ polystyrene plate.

Add 39 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 200 pg/µL.

SECTION: Tagmentation and enrichment PCR
10. This step can be carried out either by using the commercially available Nextera XT kit or an in-house Tn5 transposase, as described below. Indexing primers can be purchased from Illumina
(Nextera XT index kit v2) or ordered from your local oligonucleotide manufacturer. 
Indexing primer sequences can found in the "Materials" section.

 

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

 
Prepare the tagmentation mix:
 
            Reagent  Volume  Final   Concentration  Volume   96 rxn    Dimethylformamide   (DMF) (100%)  0.8  20%  88.32    TAPS-Mg   buffer (5x) pH 7.3  0.8  10mM   TAPS, 5mM MgCl2  88.32    Tn5   transposase (2μM working    dil.)  0.1  62.5 nmol  11.04    Nuclease-free   water  1.3     143.52    Total  3     331.2  
 
 

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

Add 1 µL normalised cDNA (200 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 °Chold. 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.
 
            Reagent  Per   sample (uL)  Final   Concentration  Volume   96 rxn    KAPA   HiFi enzyme (1U/uL) - non Hot-Start  0.2  0.02 U/μL  22.08    dNTPs   (10 mM)  0.3  300 nM  33.12    KAPA   HiFi Buffer (5x)  2  1X  220.8    Nuclease-free   water  0.5     55.2    Total   volume (μL)  3     331.2  
Seal the plate, vortex, spin down (30 s,  ), 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. 

            Step  Temperature  Time  Cycles    Gap-filling  72 °C  3 min  1x    Initial   denaturation  98°C  30s  1x    Denaturation  98°C  10s    x14      Annealing  55 °C  30 sec    Elongation  72 °C  30 sec    Final   Elongation  72 °C  5 min  1x    Hold  4 °C  Hold     


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

Remove the Sera-Mag SpeedBeads™ working solution (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 0.8X volume ratio Sera-Mag SpeedBeads™ working solution to the pool. Mix thoroughly by pipetting or vortexing.

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 minoff the magnetic stand.

Place the tube back on the magnetic stand and incubate for2 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: Sequencing
12. The clean library pool can be sequenced on any Illumina sequencer. Follow the specifications reported for each individual instrument. Single-End 75 bp is generally sufficient but longer read modes or paired-end sequenced are also options depending on the biological question.
SECTION: Analysis
13. Data generated with FLASH-seq bulk can be analysed with standard unstranded bulk RNA-sequencing pipelines. We suggest the following tools:

1. (optional) Trim left-over adapters / TSO / oligo-dT with trimmomatic or bbduk.
2. Map reads with STAR.
3. Select properly mapped reads with samtools (-F 260). 
4. Visualise the alignment with IGV.
5. Explore the mapping statistics with rseqc (gene body coverage, intron/exon/intergenic mapping).
6. Assign reads to features with featureCounts.
7. Explore isoforms with RSEM or BRIE.
8. Calculate differential expression with DESeq2, EdgeR or limma.