Single coacervate sequencing

Franziska  Aron; Damian  Wollny

Mar 07, 2022

Single coacervate sequencing

DOI

dx.doi.org/10.17504/protocols.io.bux5nxq6

Franziska Aron¹,
Damian Wollny¹

¹Friedrich-Schiller Universität Jena

Damian Wollny: Group Leader Wollny Lab

Damian DW Wollny

DOI: dx.doi.org/10.17504/protocols.io.bux5nxq6

Protocol Citation: Franziska Aron, Damian Wollny 2022. Single coacervate sequencing . protocols.io https://dx.doi.org/10.17504/protocols.io.bux5nxq6

Manuscript citation:

https://www.biorxiv.org/content/10.1101/2021.03.08.434405v1.abstract

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: May 12, 2021

Last Modified: March 07, 2022

Protocol Integer ID: 49885

Keywords: Single-cell sequencing, Liquid-liquid phase-separation, Coacervates

Disclaimer

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Abstract

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

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

Materials

AB
Primer namePrimer sequence
Oligo-dT5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3′
TSO5′-biotin- AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′
ISPCR primer5′-AAGCAGTGGTATCAACGCAGAGT-3′
All our primers were ordered from IDT.


Reagent1M MgCl2AmbionCatalog #AM9530G  
ReagentBetaine solution (5M PCR Reagent)Sigma – AldrichCatalog #B0300 
ReagentDNase I recombinant, RNase-freeSigma AldrichCatalog #000000004716728001  
ReagentdNTP Mix (dATP, dCTP, dGTP, and dTTP, each at 10mM) Thermo Fisher ScientificCatalog #R0192  
Reagenttwin.tec 96-well DNA LoBind PlatesEppendorfCatalog #0030129504  
ReagentTwin.Tec PCR Plate 96 semi-skirted, colourless wells, 25 pcsEppendorfCatalog #0030128575  
ReagentEppendorf twin.tec® PCR plate 96 LoBindEppendorfCatalog #0030129512  
Reagent2x Kapa HiFi Hotstart Readymix Kapa BiosystemsCatalog #KK2602  
ReagentNuclease-Free Water (not DEPC-Treated)Thermo FisherCatalog #AM9938  
ReagentRecombinant RNAse InhibitorTakarabioCatalog #2313A  
ReagentAgencourt RNAClean XP BeadsBeckman CoulterCatalog #A63987  
ReagentRNase ZapSigma AldrichCatalog #R2020-250ML  
ReagentSuperscript IIInvitrogen - Thermo FisherCatalog #18064-014  (Incl. 0.1M DTT and 5x FS Buffer)
ReagentTrizma hydrochloride solutionSigma AldrichCatalog #T2694  (pH 8, 1M)
ReagentEthanol (100%, Molecular Biology Grade)Fisher ScientificCatalog #BP2818500  
ReagentPoly(Diallyl Dimethyl Ammonium Chloride) [Mw ~ 8500] 28 wt. % H2OPolysciences IncCatalog #24828-100   
ReagentCM- Dextran NatriumsalzMerck Millipore SigmaCatalog #86524-10G-F  
ReagentGuanidine hydrochloride for molecular biology >=99%Sigma AldrichCatalog #G3272-500g  
ReagentNextera XT DNA Library Preparation KitilluminaCatalog #FC-131-1096 
 ReagentNextera XT Index Kit v2 (set A B C D)illuminaCatalog #FC-131-2001; FC-131-2002; FC-131  
ReagentQubit&trade; 1X dsDNA HS Assay KitThermo FisherCatalog #Q33231  
ReagentQubit RNA HS Assay KitThermo Fisher ScientificCatalog #Q32852  
ReagentQubit® Assay TubesLife TechnologiesCatalog #Q32856  


Equipment
Invitrogen™ Qubit™ 3 Fluorometer
NAME
highly sensitive fluorescence-based Qubit quantitation assays
TYPE
Invitrogen
BRAND
15387293
SKU
https://www.fishersci.de/shop/products/qubit-3-0-fluorometer/15387293
LINK
Invitrogen™ Q33216
SPECIFICATIONS

Equipment
2100 Bioanalyzer Instrument
NAME
Sizing, quantification, and sample quality control of DNA, RNA, and proteins on a single platform
TYPE
Agilent Technologies
BRAND
G2939BA
SKU

Equipment
Vortexer
NAME
VWR
BRAND
97043-562
SKU

Equipment
Centrifuge
NAME
Benchtop Centrifuge
TYPE
Eppendorf
BRAND
5405000441
SKU
https://online-shop.eppendorf.us/US-en/Centrifugation-44533/Centrifuges-44534/Centrifuge-5425-PF-243560.html
LINK
Any benchtop centrifuge will suffice
SPECIFICATIONS

Equipment
Mini-centrifuge
NAME
Centrifuge
TYPE
Fisher
BRAND
S67601B
SKU
https://www.fishersci.com/shop/products/fisherbrand-standard-mini-centrifuge-standard-mini-centrifuge/s67601b
LINK
Any standard mini centrifuge with adapters for different tube sizes will suffice
SPECIFICATIONS

Safety warnings

Ethanol
- H225 Highly flammable liquid and vapour.
- H319 Causes serious eye irritation.

Polydiallyldimethylammonium chloride
- H410 Very toxic to aquatic organisms with long lasting effects
- H412 Harmful to aquatic organisms, with long lasting effects.
  

Guanidinium Hydrochlorid
- H302 Harmful if swallowed.
- H332 Harmful if inhaled.
- H315 Causes skin irritation.
- H319 Causes serious eye irritation.


Kits
Check manufacturer's safety information for the Nextera XT Library Prep Kit used in this protocol.

Check manufacturer's safety information for the Qubit RNA HS / Qubit DNA HS Kits used in this protocol.

Check manufacturer's safety information for the RNase Away used in this protocol.

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).

Buffer preparation

Safety information
Prepare all buffers under the conditions of usage for RNA. So work RNase free. 

Prepare  Concentration6 Molarity (M)   Guanidinium hydrochloride

Prepare the droplet buffer consisting of Concentration10 millimolar (mM)  Tris and Concentration4 millimolar (mM)  MgCl2 Ph8  

Droplet production

Calculate the droplet production according to your experiment. 

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

Safety information
Droplet handling only in LoBind tubes! 


ABCDE
1000µl DropletsStock concentrationFinal concentration
Reagentsg/molMmMµl use
CM-Dextran Sodium Salt162.1416060
PDDA1741105.8
ng/µlng/µlµl use
RNA12435040.2
Droplet Buffer10mM Tris, 4mM MgCl2, pH 8893.7

Note
We measure the concentration of RNA with the Qubit RNA HS Kit 

Calculate how much RNA you need to add to the entire solution for a final concentration ofConcentration50 ng/µl   
Note
This example is made for Amount1000 µL   of droplets with a RNA stock concentration ofConcentration1243 ng/µl   

Mix the droplet buffer with the CM-Dextran Sodium Salt

Add the RNA and mix briefly

Finally add the PDDA [=Poly-{diallyl-dimethylammoniumchlorid}-solution] and mix by vortexing


Note
The solution should be turbid now 

 Add 4µl of  Concentration6 Molarity (M)   Guanidinium hydrochloride into each well of the full skirted 96-well LoBind plate 

Perform a droplet sorting via FACS into the 96-well plate. Store the sorted droplets immediately at Temperature-80 °C  or directly continue with the protocol. 

Safety information
The droplets got sorted by the FACS Facility. 

Make sure to get a positive control with 1000 droplets and also a negative control without any droplet. 

A full skirted plate usually gets recommended by the FACS facilty.

Smart-seq2 preparation

Prepare Master Mix I 


ABC
ReagentsStock concentration1x [µl]
oligo-dT primer10 µM1
dNTPs 10 mM each1
nuclease free H2O2
MasterMix Total [µl]4
Assay Total [µl]4
add all the reagents, mix by flicking the tube and spin down

Prepare Master Mix II

ABC
ReagentsStock concentration1x [µl]
nuclease free H2O0.09
MgCl21 M0.06
Betaine5 M2
DTT100 mM0.5
5xI FS Buffer5x2
RNAse inhibitor40 U/µl0.25
Superscript II RT200 U/µl0.5
TSO100 µM0.1
MM total [µl]5.5
Assay total [µl]9.5
add all the reagents, mix by flicking the tube and spin down

Note
RNAse Inhibitor, SuperScript II and TSO are sensitive reagents, add them only shortly before using the master mix

Avoid unnecessary defrosting 

Droplet clean up

Mix the amount of droplets with RNA XP beads in a 1:2.2 ratio

Vortex and spin down briefly

Note
Preheat the thermocycler to Temperature72 °C  

Depending on the experiment working TemperatureOn ice   is required. 

Incubate for Duration00:05:00   at TemperatureRoom temperature  

Pellet the beads on a magnet until solution is clear (~Duration00:05:00  )

Discard supernatant
Note
If you are losing beads during this step, leave some supernatant with beads inside the tube, rather than losing some beads

Add Amount180 µL    of 80% EtOH to the bead pellet

Discard EtOH

Add Amount160 µL    of 80% EtOH to the bead pellet

Discard EtOH
Note
The volume might change, depending on the starting volume

Remove the EtOH completely 

Resuspend the pellet in Amount4 µL   of Master Mix I by pipetting up and down

(The plate is not on the magnet for elution)
Safety information
Do not let the pellet dry. This decreases the output drastically

Smart-seq2

Incubate at Temperature72 °C   forDuration00:03:00   
Note
Finish Master Mix II during this incubation time [Go togo to step #6  ]

Spin down the reaction tube

Add Amount5.5 µL   of Master Mix II, mix by flicking the tube, spin down and start the following incubation

ABC
Cycle [Amount]Temp [°C]Time [min]
14290
10502
422
17015
4Hold

Note
Take reagents for Master Mix III out of the freezerDuration00:30:00   before the incubation time ends.

Prepare Master Mix III


ABC
ReagentsStock concentration1x [µl]
KAPA HiFi HS ReadyMix 2 x12.5
IS PCR primer10 µM0.25
nuclease free H2O2.25
MM total [µl]15
Assay total [µl]24.5
 
Mix by flicking the tube and spin down

Add Amount15 µL   of Master Mix III to the reaction 

Mix by flicking the tube and spin down

Start the following incubation
ABC
Cycle [Amount]Temp [°C]Time 
1983 min
12 - 239820 sec
6715 sec
726 min
1725 min
4Hold

Note
The amount of cycles can be varied. For single coacervate sequencing we have used 23 cycles.

Add SPRIselect beads in a 1:0.7 ratio, vortex and spin down briefly
Note
Bring the SPRIselect beads to TemperatureRoom temperature   and vortex properly before usage

Incubate for Duration00:05:00   at TemperatureRoom temperature  

Pellet the beads on a magnet until solution is clear (~Duration00:05:00  )
Discard supernatant
Note
If you are losing beads during this step, leave some supernatant with beads inside the tube, rather than losing some beads.

Add Amount180 µL   of 80% EtOH (= Ethanol, molecular biology grade) to the bead pellet

Discard EtOH

Add Amount160 µL   of 80% EtOH to the bead pellet

Discard EtOH

Remove the EtOH completely 

Resuspend the pellet in Amount17.5 µL   nuclease free H2O by pipetting up and down
(The plate is not on the magnet for elution)

Transfer the eluate into a fresh 96-well plate

Store at Temperature-20 °C  until further usage

First Quality control

Quality Control

First measure Amount1 µL   of cDNA with the Qubit HS DNA Kit according to the manufacturer's protocol

Use Amount1 µL  -Amount2 µL   of the cDNA and load it on Tapestation/Bioanalyzer

Representative Bioanalyzer trace of amplified cDNA prepared from a single coacervate

Sample: cDNA of a single droplet ; Kit: Bioanalyzer HS 2100 Expert

Tagmentation

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

Predilute cDNA to a final concentration of Concentration0.1 ng/µl    to Concentration0.3 ng/µl   in nuclease free H2O 


Note
For single-droplet experiments or negative controls we use Amount1 µL  by standard, in case of positive controls the input quantity has to be adapted if necessary.

Prepare tagmentation pre-mix as described in the following table

AB
Reagent1x (µL)
Tagment DNA buffer2.5
Amplicon Tagmentation mix (Tn5)1.25
MM total (µL)3.75

Mix by vortexing and spin down briefly

Mix tagmentation pre-mix with pre-diluted cDNA as described following

AB
Reagent1x (µL)
tagmentation pre-mix3.75
pre-diluted cDNA1.25
Assay total [µl]5

Incubate in a thermocycler as described following
ABC
Cycle [Amount]Temp [°C]Time [min]
15510
110Hold

Spin down briefly

Add Amount1.25 µL   NT buffer to each reaction, 

Mix by vortexing and spin down briefly

Tagmentation_Indexing

Note
Latest possibility to decide for indexes! 


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

AB
Reagents1x (µL)
Index primer 11.25
Index primer 21.25
Nextera PCR Master mix3.75
MM total [µl]6.25
Assay total [µl]12.5

Mix by flicking the tube and spin down

Start incubation in a thermocycler as described following
ABC
Cycle [Amount]Temp [°C]Time 
1723 min
19530 sec
129510 sec
5530 sec
7260 sec
1725 min
110hold

First bead clean up

10m

Pool all the samples

Note
Vortex the SPRIselect beads carefully before use

Add SPRIselect beads in a 1:1 ratio, mix by vortexing    

Incubate on a rotator forDuration00:05:00   at TemperatureRoom temperature  

Pellet the beads on a magnet until the solution is clear (~Duration00:05:00   )

Discard the supernatant

Wash pellet with fresh 80% EtOH p.a.

Discard the supernatant

Repeat for a total of two washes

Remove the remaining EtOH

WaitDuration00:00:30   to Duration00:01:00   

1m 30s

Elute in 1/5 of the sample volume in nuclease free H2O
Incubate for Duration00:05:00   at TemperatureRoom temperature  
(The incubation tube is not on magnet for elution)

Incubate on a magnet until the solution is clear (~Duration00:05:00   )


Note
Prepare a fresh tube with SPRIselect beads in a 1:0.8 ratio

Second bead clean up

21m 30s

Transfer the supernatant into the prepared SPRIselect beads (1:0.8), mix by vortexing

Incubate on a rotator forDuration00:05:00   at TemperatureRoom temperature  

Pellet the beads on a magnet until the solution is clear (~Duration00:05:00   )

Discard the supernatant

Wash pellet with fresh 80% EtOH 

Discard the supernatant

Repeat for a total of two washes

Remove the reminaing EtOH

Wait Duration00:00:30   to Duration00:01:00  

1m 30s

Elute in 1/5 of the sample volume in nuclease free H2O

Incubate for Duration00:05:00   atTemperatureRoom temperature  

Incuabte on a magnet until the solution is clear (~Duration00:05:00   )

Transfer the clear supernatant to a fresh tube

Store atTemperature-20 °C  until further usage

Second Quality control

Quality control

First measure Amount1 µL   of cDNA with the Qubit HS ds Kit according to the manufacturer's protocol

Use Amount1 µL  -Amount2 µL   of the cDNA and load it on Tapestation/Bioanalyer

Sample: Pool of an entire plate (1 positive control; 1 blank, 94 single droplets); Kit: Bioanalyzer HS 2100 Expert

	A	B
	Primer name	Primer sequence
	Oligo-dT	5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3′
	TSO	5′-biotin- AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′
	ISPCR primer	5′-AAGCAGTGGTATCAACGCAGAGT-3′

A	B	C	D	E
1000µl Droplets	Stock concentration		Final concentration
Reagents	g/mol	M	mM	µl use
CM-Dextran Sodium Salt	162.14	1	60	60
PDDA	174	1	10	5.8


		ng/µl	ng/µl	µl use
RNA		1243	50	40.2
Droplet Buffer	10mM Tris, 4mM MgCl2, pH 8			893.7

A	B	C
Reagents	Stock concentration	1x [µl]
oligo-dT primer	10 µM	1
dNTPs	10 mM each	1
nuclease free H2O		2
MasterMix Total [µl]		4
Assay Total [µl]		4

A	B	C
Cycle [Amount]	Temp [°C]	Time [min]
1	42	90
10	50	2
10	42	2
1	70	15
	4	Hold

A	B	C
Cycle [Amount]	Temp [°C]	Time
1	98	3 min
12 - 23	98	20 sec
	67	15 sec
	72	6 min
1	72	5 min
	4	Hold

	A	B
	Reagent	1x (µL)
	Tagment DNA buffer	2.5
	Amplicon Tagmentation mix (Tn5)	1.25
	MM total (µL)	3.75

	A	B
	Reagent	1x (µL)
	tagmentation pre-mix	3.75
	pre-diluted cDNA	1.25
	Assay total [µl]	5

	A	B
	Reagents	1x (µL)
	Index primer 1	1.25
	Index primer 2	1.25
	Nextera PCR Master mix	3.75
	MM total [µl]	6.25
	Assay total [µl]	12.5

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Single coacervate sequencing