TITLE:     Single coacervate sequencing 
AUTHORS: Franziska   Aron, Damian  Wollny

[DESCRIPTION]
Here, we present a protocol which enables the comprehensive characterization of the RNA content of single phase-separated coacervates. We adapted single-cell RNA sequencing technology in combination with fluorescence activated cell sorting (FACS)  to answer the question of how one condensate differs from the other in terms of RNA composition and how it relates to condensate features such as droplet size. This approach represents a powerful addition to labor intensive and low throughput microscopy approaches which have been the state of the art approach for coacervate RNA characterization. This protocol includes droplet production, as well as a Smart-seq2 protocol adaption for lysis, reverse transcription and cDNA amplification and sequencing library preparation. This protocol ends with the library preparation. Afterwards it got sequenced on an Illumina NextSeq500 (paired end for 300 cycles).
The Smart-seq2 protocol was originally published in Picelli, S., Faridani, O., Björklund, Å. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc9, 171–181 (2014). https://doi.org/10.1038/nprot.2014.006



[BEFORE_START]
- Take the Agencourt RNAClean XP beads out of the fridge and let them adjust to room temperature (=RT), aliquot 110µl in each tube of a 8-PCR-stripe, vortex in between to keep the beads in solution
- Defrost the reagents for the Master Mix I and Master Mix II (except enzymes and TSO)
- Unpack RNase free filter tips (10x of 10µl filter tips; 10x 200µl filter tips; 5x 20µl filter tips)
- Prepare fresh 80% EtOH p.a. (= 50ml per plate)
- Get a box full of ice

This protocol is designed for a 96-well plate (LoBind).

[GUIDELINES]
Follow the general guidelines for working with RNA

- wear clean gloves all the time
- use sterile equipment and sterile disposable plasticware
- use a designated area for RNA work only
- use RNase inactivating reagents to clean equipment and surfaces
- Use RNase/DNase free filter tips instead of normal tips
- work preferably quick and on ice
- avoid handling over open bottles/tubes/etc.
- avoid RNase contamination through air
- use RNase free water

- RNA storage temperature is -80°C, avoid defrosting cycles

[STEPS]
SECTION: Droplet production
4. 
Calculate the droplet production according to your experiment. 

The example is made for CM-Dextran:PDDA coacervates (molar ratio: 6:1) 

 


 

 

SECTION: Droplet production
4.2. Mix the droplet buffer with the CM-Dextran Sodium Salt
SECTION: Droplet production
4.3. Add the RNA and mix briefly


SECTION: Droplet production
4.4. 
Finally add the PDDA [=Poly-{diallyl-dimethylammoniumchlorid}-solution] and mix by vortexing


 

SECTION: Droplet production
4.1. Calculate how much RNA you need to add to the entire solution for a final concentration of50 ng/µl 
 


SECTION: Smart-seq2 preparation
5. Prepare Master Mix I 


 

SECTION: Smart-seq2 preparation
6. Prepare Master Mix II

 

 

SECTION: Droplet clean up
7. 
Mix the amount of droplets with RNA XP beads in a 1:2.2 ratio

Vortex and spin down briefly

SECTION: Droplet clean up
7.2. Incubate for 5 min at 72 Room temperature

SECTION: Droplet clean up
7.5. Add 160 µL  of 80% EtOH to the bead pellet

Discard EtOH
 

SECTION: Droplet clean up
7.3. Pellet the beads on a magnet until solution is clear (~5 min)

Discard supernatant
 

SECTION: Droplet clean up
7.4. Add 180 µL  of 80% EtOH to the bead pellet

Discard EtOH


    
SECTION: Droplet clean up
7.7. Resuspend the pellet in 4 µL of Master Mix I by pipetting up and down

(The plate is not on the magnet for elution)
 

SECTION: Droplet clean up
7.6. Remove the EtOH completely 
SECTION: Droplet production
4.6. Perform a droplet sorting via FACS into the 96-well plate. Store the sorted droplets immediately at -80 °Cor directly continue with the protocol. 


 


SECTION: Smart-seq2
9. Spin down the reaction tube

Add 5.5 µL of Master Mix II, mix by flicking the tube, spin down and start the following incubation




 


 
 


SECTION: Smart-seq2
10. Prepare Master Mix III


 
 
Mix by flicking the tube and spin down
9.1. 
SECTION: Smart-seq2
10.1. Add 15 µL of Master Mix III to the reaction 

Mix by flicking the tube and spin down
SECTION: Smart-seq2
10.2. Start the following incubation
 

 

SECTION: Smart-seq2
11. Add SPRIselect beads in a 1:0.7 ratio, vortex and spin down briefly
 



SECTION: Smart-seq2
11.1. Incubate for 5 min at Room temperature

SECTION: Smart-seq2
11.2. Pellet the beads on a magnet until solution is clear (~5 min)
Discard supernatant
 

SECTION: Smart-seq2
11.3. Add 180 µL of 80% EtOH (= Ethanol, molecular biology grade) to the bead pellet

Discard EtOH


SECTION: First Quality control
12. Quality Control

First measure 1 µL of cDNA with the Qubit HS DNA Kit according to the manufacturer's protocol

SECTION: Smart-seq2
11.6. Resuspend the pellet in 17.5 µL nuclease free H2O by pipetting up and down
(The plate is not on the magnet for elution)
SECTION: Smart-seq2
11.5. Remove the EtOH completely 
SECTION: Smart-seq2
11.4. Add 160 µL of 80% EtOH to the bead pellet

Discard EtOH


SECTION: Smart-seq2
11.8. Store at -20 °Cuntil further usage

SECTION: First Quality control
12.1. Use 1 µL-2 µL of the cDNA and load it on Tapestation/Bioanalyzer

Representative Bioanalyzer trace of amplified cDNA prepared from a single coacervate


  



SECTION: Smart-seq2
8. Incubate at 72 °C for3 min 
 



SECTION: Tagmentation
13. Get a box full of ice, bring Tagment DNA buffer and NT buffer (Illumina, Nextera XT Library Prep Kit) to room temperature

Decide already about the index combinations that you want to use

SECTION: Tagmentation
14. Predilute cDNA to a final concentration of 0.1 ng/µl  to 0.3 ng/µl in nuclease free H2O 


 

SECTION: Tagmentation
15. Prepare tagmentation pre-mix as described in the following table

 

Mix by vortexing and spin down briefly
SECTION: Tagmentation
17. Incubate in a thermocycler as described following
 

SECTION: Tagmentation
18. Spin down briefly

Add 1.25 µL NT buffer to each reaction, 

Mix by vortexing and spin down briefly

SECTION: Tagmentation_Indexing
19. 
 


Prepare the indexing reaction as described in the following table and add to the reaction

 

Mix by flicking the tube and spin down
SECTION: Tagmentation_Indexing
20. Start incubation in a thermocycler as described following
 



SECTION: First bead clean up
21. Pool all the samples

 

Add SPRIselect beads in a 1:1 ratio, mix by vortexing    
SECTION: First bead clean up
22. Incubate on a rotator for5 min at Room temperature


SECTION: First bead clean up
23. Pellet the beads on a magnet until the solution is clear (~5 min )

Discard the supernatant
SECTION: First bead clean up
24. Wash pellet with fresh 80% EtOH p.a.

Discard the supernatant




SECTION: First bead clean up
25. Repeat for a total of two washes

SECTION: First bead clean up
26. Remove the remaining EtOH

Wait30 s to 1 min 

SECTION: First bead clean up
27. Elute in 1/5 of the sample volume in nuclease free H2O
Incubate for 5 min at Room temperature
(The incubation tube is not on magnet for elution)

SECTION: First bead clean up
28. Incubate on a magnet until the solution is clear (~5 min )


 

SECTION: Second bead clean up
29. Transfer the supernatant into the prepared SPRIselect beads (1:0.8), mix by vortexing


SECTION: Second bead clean up
30. Incubate on a rotator for5 min at -20 Room temperature

SECTION: Second bead clean up
31. Pellet the beads on a magnet until the solution is clear (~5 min )

Discard the supernatant
SECTION: Second bead clean up
32. Wash pellet with fresh 80% EtOH 

Discard the supernatant

SECTION: Second bead clean up
33. Repeat for a total of two washes
SECTION: Second bead clean up
34. Remove the reminaing EtOH

Wait 30 s to 1 min

SECTION: Second bead clean up
35. Elute in 1/5 of the sample volume in nuclease free H2O

Incubate for 5 min at-20 Room temperature

SECTION: Second bead clean up
36. Incuabte on a magnet until the solution is clear (~5 min )

Transfer the clear supernatant to a fresh tube
SECTION: Second bead clean up
37. Store at-20 °Cuntil further usage

SECTION: Second Quality control
38. Quality control

First measure 1 µL of cDNA with the Qubit HS ds Kit according to the manufacturer's protocol
SECTION: Buffer preparation
3. Prepare the droplet buffer consisting of 10 Milimolar (mM)Tris and 4 Milimolar (mM)MgCl2 pH 8 




SECTION: Tagmentation
16. Mix tagmentation pre-mix with pre-diluted cDNA as described following

 

SECTION: Second Quality control
39. Use 1 µL-2 µL of the cDNA and load it on Tapestation/Bioanalyer

  

SECTION: Droplet production
4.5.  Add 4µl of  6 Molarity (M) Guanidinium hydrochloride into each well of the full skirted 96-well LoBind plate 

SECTION: Droplet clean up
7.1. 
 

SECTION: Buffer preparation
2. Prepare  6 Molarity (M) Guanidinium hydrochloride



SECTION: Smart-seq2
11.7. Transfer the eluate into a fresh 96-well plate
SECTION: Buffer preparation
1.