TITLE: MINECRAFTseq V1
AUTHORS: Yuriy Baglaenko

[DESCRIPTION]
Genetic studies have identified thousands of individual disease-associated non-coding alleles, but identification of the causal alleles and their functions remain critical bottlenecks. Even though CRISPR-Cas editing has enabled targeted modification of DNA, inefficient editing leads to heterogeneous outcomes across individual cells, limiting the ability to detect functional consequences of disease alleles. To overcome these challenges, we present a multi-omic single cell sequencing approach that directly identifies genomic DNA edits, assays the transcriptome, and measures cell surface protein expression. We apply this approach to investigate the effects of gene disruption, deletions in regulatory regions, and non-coding single nucleotide polymorphisms. 

[STEPS]
SECTION: Lysis plate generation
1. In a PCR clean hood, prepare a lysis mix containing the following reagents: 
 

 

Seal the plates with an aluminum PCR seal and spin down for 1 minute at 1000g, 4°C.

Proceed immediately to the next step or store the plate on ice to avoid denaturing of Recombinant RNase Inhibitor.
For long-term storage, store the plates in -20 °C. 
SECTION: Single cell sorting
2. Sort single cells into wells containing 1 µL of the lysis buffer. 
Cells can be stained with fluorophore-conjugated and oligoconjugated antibodies. Indexing flow cytometry can be used to collect this information to hash multiple conditions. 
This can be achieved in any number of ways. We routinely use the BIGFOOT from Thermofisher for rapid and accurate sorting into 384 well plates. 
We have discovered that staining with PI or 7-AAD seems to interfere with downstream applications. We would not recommend doing this. 

Immediately after sorting, seal the plate with an aluminum PCR seal and spin down for 1 minute at 1000g, 4 °C. 

Place the plate containing single cells on dry ice if processing multiple plates until ready to transfer them all into a -80 °C freezer.
SECTION: RNA denaturation
3. Thaw the plates containing single cells at room temperature for 1 minute.

Make sure the aluminum PCR seal is still intact. Re-seal if necessary. 

Incubate the plate in a thermocycler with a heated lid for 3 minutes at 72°C, followed by a 4 °C hold step.
SECTION: RT-PCR
4. 
 


Prepare the following RT-PCR master mix:
These volumes account for a 15% pipette error. 
 

Add 4 µL of the master mix into each well of the 384-well plate. Seal with an aluminum PCR seal and proceed to RT incubation. This can be done with any number of liquid handling/dispensing systems. In our hands, both the Bravo and MANTIS systems have been applied to distribute master mixes. 

In a thermocycler with a heated lid, start the RT step:
 
After the RT step, remove the plate from the thermocycler and place it on ice.

Add primers for amplification of genomic DNA and ADT:
 

 

Seal the plate with an aluminum PCR seal and spin down for 1 minute at 1000g, 4 °C. 

In a thermocycler with a heated lid, start the following PCR program: 
 

 


Store plates at -20 °C until continuing with nested genomic DNA, pooling, and cleanup.
SECTION: Nested genomic DNA PCR
5. In a sterile hood, prepare a PCR master mix using the following reagents:
These volumes account for a 15% pipette error.
 
Add 3.6 µL of the master mix to each well in a clean 384-well plate.

Seal the plate with an aluminum PCR seal and spin down for 1 minute at 1000g, 4 °C. 

Add 0.4 µL of barcoded capture primers to each well.  

 

Thaw the pre-amplified RT-PCR plate from -20 °C. Making sure that the aluminum seal is still intact, spin down for 1 minute at 1000g, 4 °C.

Aliquot 1 µL of the pre-amplified RT-PCR plate into the 384-well plate containing the master mix to perform the nested PCR step. 

 

Store the pre-amplified RT-PCR plate at -20 °C as it will be used in step 7 (cDNA Library Preparation).

Seal the plate containing the master mix with an aluminum PCR seal and proceed to the PCR step:
 
 
After the PCR step, proceed to pool the samples and SPRI clean of genomic DNA or store at -20 °C.
SECTION: Genomic DNA Library Preparation
6. 
 


DNA Pooling:
If the plate was stored at -20°C, thaw for 1 minute.
After making sure that the aluminum seal is still intact, spin down for 1 minute at 1000g, 4°C.
Pool 2ul of the product from each well into a clean 1.5ml tube. This can be performed on a Bravo liquid handling platform. 


SPRI clean-up:
From the pool, take 315ul for SPRI clean-up and store the rest at -20°C. SPRI clean at 1.2X. 
To 315ul of product, add 378ul of room temperature SPRI.
Incubate at room temperature for 10 minutes.
Place the tube on a magnet for 5 minutes.
While keeping the tube on the magnet, discard the supernatant.
While keeping the tube on the magnet, wash twice with 80% freshly made ethanol. Wait for 30 seconds. Remove ethanol carefully without touching the product.
While keeping the tube on the magnet, dry the tube for 10-15 minutes.
Take the tube off the magnet and resuspend in 40ul of Nuclease-free water. Incubate for 2-5 minutes.
Place the tube on the magnet for 5 minutes.
While keeping the tube on the magnet, transfer 37ul of the supernatant to a new tube. This is the gDNA amplicon. 
      
Measure DNA concentration on a QuBit. Proceed to ExoI and ExoVII treatments and P5-P7 amplification reaction. 
There is often no point in checking distribution at this point because the product might not be detectable. 


ExoVII treatment:
This step is done to remove the excess capture primers and OligoDT.
Dilute the gDNA amplicons to 2ng/ul in Nuclease-free water.
Use 10ul of the 2ng/ul gDNA amplicon to perform ExoI nuclease reactions in PCR plates.
Prepare a master mix containing the following reagents: SOMEONE DOUBLECHECK THESE CALCULATIONS AND REAGENTS
 
 Seal the plate containing the master mix with an aluminum PCR seal and follow the following steps using a thermocycler:
 


re-SPRI: 
This step is done to remove the ExoVII nuclease. Take 20ul for SPRI clean-up and store the rest at -20°C. SPRI clean at 1.2X. 
To 20ul of product, add 24ul of room temperature SPRI.
Incubate at room temperature for 10 minutes.
Place the tube on a magnet for 5 minutes.
While keeping the tube on the magnet, discard the supernatant.
While keeping the tube on the magnet, wash twice with 80% freshly made ethanol. Wait for 30 seconds. Remove ethanol carefully without touching the product.
While keeping the tube on the magnet, dry the tube for 10-15 minutes.
Take the tube off the magnet and resuspend in 11ul of Nuclease-free water. Incubate for 2-5 minutes.
Place the tube on the magnet for 5 minutes.
While keeping the tube on the magnet, transfer 10ul of the supernatant to a new plate for immediate P5-P7 amplification reaction. 


P5-P7 Amplification:
Prepare the following master mix for the P5-P7 amplification reaction:
To 10ul of gDNA amplicon, add 10ul of the master mix without the P5 and P7 primers.
Add 2.5ul of the P5 primer and 2.5ul of the unique P7 primer and mix.
 

 

Seal the plate with an aluminum PCR seal and proceed to the amplification reaction in a thermocycler:
 
 
Pool the products from the amplification reaction and proceed to the last DNA SPRI clean-up step. HOW MUCH TO POOL


Final SPRI clean-up:
SPRI clean at 1.2X. 
Example: 
To 25ul of product, add 30ul of room temperature SPRI.
Incubate at room temperature for 10 minutes.
Place the tube on a magnet for 5 minutes.
While keeping the tube on the magnet, discard the supernatant.
While keeping the tube on the magnet, wash twice with 80% freshly made ethanol. Wait for 30 seconds. Remove ethanol carefully without touching the product.
While keeping the tube on the magnet, dry the tube for 10-15 minutes.
Take the tube off the magnet and resuspend in 10ul of Nuclease-free water. Incubate for 2-5 minutes.
Place the tube on the magnet for 2 minutes.
Save the eluent - this is the DNA P5/P7.

Spec on a Qubit. Proceed to check on a Tape Station. (EXAMPLE PHOTO HERE)

Proceed to sequencing.

 

SECTION: cDNA Library Preparation
7. 
 


cDNA Pooling:
Thaw the pre-amplified RT-PCR plate from -20°C for 1 minute at room temperature. 
Making sure that the aluminum seal is still intact, spin down for 1 minute at 1000g, 4°C.
Pool 2ul of the product from each well into a clean 1.5ml tube. This can be performed on a Bravo liquid handling platform. 
Label the tube as RT-PCR.


SPRI clean at 0.6X:
To 315ul of product, add 190ul of room temperature SPRI.
Incube at room temperature for 10 minutes.
Place the tube on a magnet for 5 minutes.
While keeping the tube on the magnet, save 300ul of the supernatant into a new tube (which will be used in step 8 ADT Library Preparation).
Discard the rest of the supernatant.
While keeping the tube on the magnet, wash twice with 80% freshly made Ethanol. Wait for 30 seconds. Remove ethanol carefully without touching the product.
While keeping the tube on the magnet, dry the tube for 10-15 minutes.
Take the tube off the magnet and resuspend in 21ul of Nuclease-free water. Incubate for 2-5 minutes.
Place the tube on the magnet for 5 minutes.
While keeping the tube on the magnet, transfer 20ul of the supernatant to a new 96-well plate. This is the full-length cDNA (which can be labeled as cDNA).

Spec on a QuBit. Check the distribution of cDNA libraries on DS5000 tape, one plate per primer. Banding is ok. (WE SHOULD INCLUDE AN EXAMPLE PHOTO HERE)


ExoI treatment:
To 18ul of the cDNA sample, add 2ul of ExoI and 5ul of Q5 (5X buffer).
Mix and incubate in a thermocycler:
 

 


re-SPRI:
Transfer to a 1.5ml tube and proceed to re-SPRI at 0.6X:
To 25ul of product, add 15ul of room temperature SPRI.
Incube at room temperature for 10 minutes.
Place the tube on a magnet for 5 minutes.
While keeping the tube on the magnet, save 300ul of the supernatant into a new tube. Discard the rest.
While keeping the tube on the magnet, wash twice with 150ul of 80% freshly made Ethanol. Wait for 30 seconds. Remove ethanol carefully without touching the product.
While keeping the tube on the magnet, dry the tube for 10-15 minutes.
Take the tube off the magnet and resuspend in 21ul of Nuclease-free water. Incubate for 2-5 minutes.
Place the tube on the magnet for 5 minutes.
While keeping the tube on the magnet, transfer 20ul of the supernatant to a new tube. This is the full-length cDNA (which can be labeled as cDNA).

Proceed to tagmentation and P5-P7 amplification reaction.


Nextera Tagmentation:
 

Dilute the full-length cDNA to 2ng/ul in Nuclease-free water.
In a 96-well PCR plate, add 5ul of Illumina Tagment DNA TDE1 buffer.
Add 2.5ul of the 2ng/ul cDNA.
Pipette to mix.
Add 2.5ul of Illumina Amplicon Tagment Mix.
Pipette to mix. Seal with aluminum PCR seal. Spin down for 1 minute at 1000g, 4°C.
Run the following program in a thermocycler:
 
Immediately when the reaction stabilizes at 10°C, add 2.5ul of Illumina Neutralize Tagment buffer.
Pipette to mix. Seal with aluminum PCR seal. Spin down for 1 minute at 1000g, 4°C.
Incubate for 7 minutes at room temperature to deactivate the Tn5.
Meanwhile, prepare the following master mix:
 

P5-P7 Amplification:
When the incubation is done, add 2.5ul of unique RNA P7 primer.
Then, add 10ul of the master mix.
Seal with aluminum PCR seal and proceed to the amplification step in a thermocycler:
 
Pool the products from the amplification reaction and proceed to the last cDNA SPRI clean-up step. HOW MUCH TO POOL


Final SPRI clean-up:
SPRI clean at 0.8X. 
Example: 
To 25ul of product, add 20ul of room temperature SPRI.
Incubate at room temperature for 10 minutes.
Place the tube on a magnet for 5 minutes.
While keeping the tube on the magnet, discard the supernatant.
While keeping the tube on the magnet, wash twice with 80% freshly made ethanol. Wait for 30 seconds. Remove ethanol carefully without touching the product.
While keeping the tube on the magnet, dry the tube for 10-15 minutes.
Take the tube off the magnet and resuspend in 10ul of Nuclease-free water. Incubate for 2-5 minutes.
Place the tube on the magnet for 2 minutes.
Save the eluent - this is the RNA P5/P7.

Spec on a Qubit. Proceed to check on a Tape Station. (EXAMPLE PHOTO HERE)

Proceed to sequencing.

 

SECTION: ADT Library Preparation
8. 
 


SPRI clean at 1.4X:
To 300ul of product, add 420ul of room temperature SPRI.
Incube at room temperature for 10 minutes.
Place the tube on a magnet for 5 minutes.
While keeping the tube on the magnet, discard the supernatant.
While keeping the tube on the magnet, wash twice with 80% freshly made Ethanol. Wait for 30 seconds. Remove ethanol carefully without touching the product.
While keeping the tube on the magnet, dry the tube for 10-15 minutes.
Take the tube off the magnet and resuspend in 40ul of Nuclease-free water. Incubate for 2-5 minutes.
Place the tube on the magnet for 5 minutes.
While keeping the tube on the magnet, transfer 37ul of the supernatant to a new tube. This is the ADT (which can be labeled as ADT).

Spec on a QuBit. Proceed to ExoI and ExoVII treatments and P5-P7 amplification reaction. 
There is often no point in checking distribution at this point because the product might not be detectable. 


ExoI treatment: DOUBLECHECK REAGENTS (IS IT JUST EXOI AND WATER + SAMPLE)
This step is done to remove the excess capture primers and OligoDT.
Dilute the ADT sample to 2ng/ul in Nuclease-free water.
Use 10ul of the 2ng/ul ADT sample to perform Exo nuclease reactions in PCR plates.
Prepare a master mix containing the following reagents: SOMEONE DOUBLECHECK THESE CALCULATIONS
 

Seal the plate containing the master mix with an aluminum PCR seal and follow the following program using a thermocycler:
 

Proceed to P5-P7 amplification. There is no need to SPRI after the ExoI reaction as the ExoI nuclease is thermolabile.


P5-P7 Amplification:
Prepare the following master mix for the P5-P7 amplification reaction:
To 10ul of the sample containing ADT, add 10ul of the master mix without the P5 and P7 primers.
Add 2.5ul of the P5 primer and 2.5ul of the unique P7 primer and mix.
 

 

Seal the plate with an aluminum PCR seal and proceed to the amplification reaction in a thermocycler:
 

Pool the products from the amplification reaction and proceed to the last ADT SPRI clean-up step. HOW MUCH TO POOL


Final SPRI clean-up:
SPRI clean at 1.6X. 
Example: 
To 25ul of product, add 40ul of room temperature SPRI.
Incubate at room temperature for 10 minutes.
Place the tube on a magnet for 5 minutes.
While keeping the tube on the magnet, discard the supernatant.
While keeping the tube on the magnet, wash twice with 80% freshly made ethanol. Wait for 30 seconds. Remove ethanol carefully without touching the product.
While keeping the tube on the magnet, dry the tube for 10-15 minutes.
Take the tube off the magnet and resuspend in 10ul of Nuclease-free water. Incubate for 2-5 minutes.
Place the tube on the magnet for 2 minutes.
Save the eluent - this is the ADT P5/P7.

Spec on a Qubit. Proceed to check on a Tape Station. (EXAMPLE PHOTO HERE)

Proceed to sequencing. 

 

9.