Cell Interaction by Multiplet sequencing (CIM-seq)

CIM-seq is compatible with both plate based and droplet based single cell RNA-seq methods. The following protocol is for Smart-seq2 based CIM-seq. For droplet based (10x), see the methods section of the manuscript and the vignettes in the Enge lab Github repository.

Prepare Lysis Buffer:

NOTE: Reagents are prepared on ice, working quickly. ERCC is stored in single-use aliquots at -80°C, thawed on ice and added last.

A	B	C
Reagent:	Reagent concentration:	μl per reaction:
H20	-	1.31
Inhibitor	1 U/μl	0.05
ERCC (1:600 000)	-	0.05
Triton-X100 (10% solution)	0.2%	0.04
10mM dNTP	2.5mM/each	0.5
100uM dT	2.5μM	0.05
Total	-	2

Add 2µL lysis buffer mix to each well. Cover with appropriate lids. Spin down.

Snap freeze on dry ice . Store until use at -80°C

Sort single cells and multiplets (aggregates of multiple cells) into 2µL lysis buffer mix.

Multiplets can be discerned from singlets by gating on the basis of FSC-W (Forward scatter - Width) and FSC-H (Forward scatter - Height) (see Figure 1 ).

Following sort, immediately seal with appropriate seals (approved for -80C > 100C) and centrifuge at 2000x g,4°C.

Snap freeze on dry ice . Store until use at -80°C .

Primer annealing

Thaw plate. Spin down. Incubate in thermocycler at 72°C for 0h 3m 0s . Place on ice immediately.

Prepare RT master-mix

Made fresh.

A	B	C
Reagent:	Reaction concentration:	Reagent volume:
SmartScribe	15u/µl	0.475
RNase Inhibitor	1.66u/µl	0.125
5x First Strand buffer	1X	1
DTT (100mM)	8.33mM	0.25
Betaine (5M) [fridge]	1.66M	1
MgCl2 (1M) [bench]	10mM	0.03
TSO (100uM)	1.66µM	0.05
H20	-	0.07
Total	-	3

Dispense 3µL per well.

Cover plate with new film and spin down.

Incubate in thermocycler

42°C 1h 30m 0s

70°C 0h 5m 0s

4°C hold

cDNA preamplification

Made fresh.

A	B	C
Reagents	Reaction concentration:	Reagent volume:
H2O	-	1.0688
Kapa HiFi HotStart ReadyMix (2x)	1X	6.25
IS_PCR primer (10uM)	0.1µM	0.125
Lambda Exonuclease	0,045u/µl	0.05625
Total	-	7.5000

Dispense 7.5µL per well . Total reaction volume will be 12.5µl.

Spin down. Cover with new lid.

Incubate in thermocycler with the following program:

A	B	C	D
Step	Temperature	Time	Cycles
Lambda exonuclease	37ºC	30 min	1x
Initial denaturation	95ºC	3 min	1x
Denaturation	98ºC	20 sec	18-24x
Annealing	67ºC	15 sec
Elongation	72ºC	4 min
Final elongation	72ºC	5 min	1x
	4C	Hold

cDNA cleanup

We prepare SPRI-beads in 20% PEG-8000 solution as in: https://openwetware.org/wiki/SPRI_bead_mix#Ingredients_for_50_mL_2

Using 20% SPRI-bead solution:

1. Add 0.7x the reaction volume of SPRI beads per well. Mix well by pipetting. (i.e 8.75µL SPRI-bead solution for 12.5µL reaction volume)

2. Incubate 0h 5m 0s Room temperature

3. Place on magnetic stand for 0h 3m 0s

4. Carefully remove supernatant

5. Add 40 µl 80% EtOH and incubate 0h 0m 30s

6. Remove EtOH (without disturbing the beads)

7. Wash again with EtOH. Make sure to remove well.

8. Allow beads to air-dry for 0h 10m 0s -0h 15m 0s

9. Remove plate from magnetic stand

10. Elute beads in 15µL EB or TE buffer. Mix well by pipetting

11. Incubate 0h 5m 0s Room temperature

12. Place on magnetic plate for 0h 3m 0s

13. Optional: Carefully remove supernatant to the elution plate

cDNA quantification

We measure concentration of random wells using Qubit HS dsDNA, adapted to a 96-well plate reader.

1. Add 97µL of 1X Qubit HS dsDNA solution to a flat-bottom, black plate

2. Add 3µL of cDNA sample

3. Add Standards (NOTE: We make a 8-step ladder from 0ng/µl --> 10ng/µl Qubit Standard DNA in TE buffer)

4. Read in plate reader using 485nM excitation/528nm emission

5. Calculate cDNA concentration

(optional) cDNA quality control

Using Agilent HS 5000 DNA chips (or equivalent)

Make cDNA dilution plate

Dilute cDNA in water based on average concentration from Qubit measurements.

Target concentration 150pg per µl in 15µL.

Tn5 digestion

Tn5 is produced from psfTn5 (Addgene #79107), purified to ~3mg/ml and assembled with Illumina Tn5 adapters (see oligos) as in Picelli et al, 2014.

Prepare Tn5 master mix

A	B	C
Reagent	Reaction conc.	µl per reaction
Nuclease free H2O	-	1.05
TAPS-PEG (50mM TAPS, 25mM MgCl2, 40% PEG-8000)	10mM TAPS, 5mM MgCl2, 8% PEG-8000	0.5
psfTn5, loaded with 50µM MEDS-A/B		0.25
Total		1.8

Dispense 1.8µL per well in a new plate(tagmentation plate)

Add 0.7µL cDNA (normalized to 150pg/µl)

Mix well by vortexing plate. Cover with new lid and spin down.

Incubate in thermocycler at 55°C``0h 10m 0s

Remove immediately and stop reaction by adding 1µL per well of 0.1% SDS.

Vortex, spin down and incubate 0h 7m 0s at 55Room temperature

Make PCR master-mix

A	B
Reagents	µl per reaction
H2O	13.25
5x buffer	5
dNTPs	0.75
KAPA	0.5
Total	19.50

Dispense 19.5µL per well to tagmentation plate (containing 3.5µL sample after step 14)

Add primers/barcodes

2µL per well (from 384-well index plates, with 3.75µM/each forward/reverse primers; see oligos in materials ).

Total reaction volume is 25µL (3.5µL sample + 19.5µL PCR mix and 2µL primers).

Vortex. Spin down and cover. Incubate in thermocycler as following:

A	B	C	D
Step	Temperature	Time	Cycles
Gap fill	72ºC	3 min	1x
First denature	95ºC	30 sec	1x
Denature	95ºC	15 sec	12x
Denature	67ºC	30 sec
Denature	72ºC	45 sec
Final extension	72ºC	4 min	1x
	4-10ºC	hold

Pool 2.5µL from each well to an 1.5ml Eppendorf tube.

Library cleanup

1. Add 0.9x pooled library volume of SPRI-bead solution. Incubate for 0h 5m 0s at Room temperature .

2. Place on magnetic rack for 0h 3m 0s.

3. Remove supernatant without disturbing magnetic beads.

4. Add at least 1mL 80% EtOH (fresh). Incubate for 0h 0m 30s.

5. Remove supernatant.

6. Repeat EtOH wash.

7. Air dry for 0h 10m 0s - 0h 15m 0s.

8. Re-suspend beads thoroughly in 100 µl EB or TE buffer.

9. Place eppendorf on magnetic rack for 0h 3m 0s.

10. Transfer supernatant to new 1.5ml Eppendorf tube.

11. Repeat cleanup (from step 1-7) and elute in 30 µl EB or TE buffer.

12.(Optional) Place eppendorf on magnetic rack for 0h 3m 0s and transfer supernatant to new tube.

Pooled library QC

A series of pre-processing steps must be performed in order to generate a counts file:

1. Trim reads, remove adapter sequences and align RNAseq data to reference genome using STAR:

2. Remove duplicate reads using Picard:

3. Generate transcript counts file using HTSeq:

Once a counts file has been generated the data can be analyzed. CIM-seq requires four arguments in order to run:

1. The raw counts data with gene IDs as rownames and sample IDs as

colnames.

2. The ERCC spike-in counts data with gene IDs as rownames and sample

IDs as colnames.

3. The dimensionality reduced representation of the data.

4. A class for each of the individual singlets.

In order to generate the last two of these we recommend using the Seurat package in R, as CIM-seq is implemented in R as well. A number of tutorials for Seurat can be found on the Satijalab website:

https://satijalab.org/seurat/vignettes.html

CIM-seq can be downloaded from:

https://github.com/EngeLab/CIMseq

Or installed directly in R using the devtools package:

devtools::install_github("EngeLab/CIMseq")

The CIM-seq vignette can be found at:

https://github.com/EngeLab/CIMseq/tree/master/vignettes