TITLE: cDNA Exome Capture v1.0.1
AUTHORS: Parse Biosciences, Elisabeth Rebboah

[DESCRIPTION]
This protocol describes a modified version of the Parse Biosciences Gene Capture protocol for full-length cDNA meant for both Illumina short read and Oxford Nanopore long read library prep and sequencing. We use Twist Biosciences exome panels to enrich for non-intronic reads for more meaningful long-read data. Each section matches the original protocol but with our modifications. Please see the attachment for the original Parse Biosciences protocol. The main deviations from the original Parse Biosciences protocol are 1. the use of cDNA amplification reagents instead of the amplification reagents included in the Gene Capture kit, and 2. modification of SPRI bead ratio from 1.8x to 0.8x. We perform this protocol on cDNA generated for a subset of 13,000 barcoded cells or nuclei from the WT or WT Mega kits.

The product of this protocol is full-length, barcoded cDNA that excludes intron-only fragments. Each molecule should have a cell/nuclei barcode, UMI, and some fraction overlapping a region found in the Twist human or mouse exome panel. Given an input of 500 ng full-length, barcoded cDNA from the end of Section 2 (WT / WT Mega protocol), there should be plenty of captured cDNA (> 1 ug). In our experience, an input as low as 200 ng resulted in 700 ng captured cDNA.

The first part of the protocol, Section 1, describes setting up hybridization repetitive sequence blockers and of the actual panel, which runs overnight. Section 2 describes capture of the hybridized molecules to streptavidin beads and amplification of the captured product. Section 2 section uses cDNA amplification reagents from the WT / WT Mega kits.

[BEFORE_START]
User Supplied Equipment and Consumables: Before starting an experiment, check the “User Supplied Equipment and Consumables” section and confirm that your lab has all of the supplies that are not provided by the kit. Avoid substituting custom materials for those that are provided in the kit. Each item has been deliberately chosen to attain optimal results.
Input:  This protocol begins with indexed sublibraries generated with an Evercode Whole Transcriptome kit. Up to 8 sublibraries can be pooled into each Gene Capture reaction. Thus, all 8 sublibraries from an Evercode WT kit can be pooled into 1 reaction, and all 16 sublibraries from an Evercode WT Mega would require 2 reactions. We recommend adding 100 ng of each sublibrary to a Gene Capture reaction for a maximum of 800 ng of input. To ensure even coverage,
sublibraries should be mixed in equal proportion. However, if any sublibraries have less than 100 ng of input, sublibraries can be pooled to:
(A) Maintain the desired proportion between samples with lower final library complexity. or (B) Add the maximum amount of lower concentration indexed
sublibraries, which will result in the highest overall complexity but impact the proportion of reads between samples.
Nonhuman Samples: If using a non-human samples, the Blocker Solution can be replaced with a species-specific blocking solution (not provided). The provided Blocker Solution may still reduce off target binding in other vertebrate species but less effectively.
Concentration Sublibraries: Sublibraries can be concentrated with vacuum centrifugation rather than SPRI bead concentration. See Appendix for a protocol.
Gene Panels: Gene Capture is compatible with the Immune1000 Panel and custom gene panels from Twist Biosciences. The Immune1000 Panel is included with Immune1000 Gene Capture (GCE1002). For Custom Gene Capture (GCE1001), a panel is not provided and should be purchased separately
from Twist Biosciences. Custom panel design can vary based on experimental goals, but we recommend that probes span the MANE transcript of each gene with non-overlapping probes, including the 3’ UTR. For additional details about panel design, contact us at support@parsebiosciences.com.

For additional questions not discussed above, please contact us at support@parsebiosciences.com. We also have a library of additional resources and videos on our support site at https://support.parsebiosciences.com/.

[STEPS]
SECTION: Section 1: Hybridization
1. 1.1 Prepare cDNA
SECTION: Section 1: Hybridization
1.1. Prepare for hybridization with the following checklist:
Fill an ice bucket
Set a heat block to 65C
Program a thermocycler to 95C and set the heated lid to 105C
Equilibrate SPRI beads (Ampure XP or KAPA Pure Beads) to room temperature for at least 30 minutes
Prepare at least 600 µL of 85% ethanol per reaction
Take out magnetic rack for 1.5 mL tubes

SECTION: Section 1: Hybridization
1.2. Gather the following items and handle as indicated below:

 

SECTION: Section 1: Hybridization
1.3. Incubate the Hybridization Mix in the heat block at 65°C for 10 minutes, or until no precipitate is present before proceeding.
SECTION: Section 1: Hybridization
1.4. Incubate the Hybridization Mix at room temperature for 5 minutes to equilibrate the solution.
SECTION: Section 1: Hybridization
1.5. Vortex cDNA (2-3 sec). Briefly centrifuge (~2 sec).
SECTION: Section 1: Hybridization
1.6. For each hybridization reaction, we recommend adding 500 ng of each cDNA sample to a Gene Capture reaction. However, we've performed gene capture and long read library prep with as low as 200 ng of cDNA input. Do NOT pool cDNA samples, they do not have the fourth subpool barcode (Illumina index)!
SECTION: Section 1: Hybridization
1.7. If the total volume of any cDNA sample is less than 100 μL, bring the volume up to 100 μL with nuclease-free water.
SECTION: Section 1: Hybridization
1.8. Vortex the SPRI beads until fully mixed. Add 0.8x of SPRI beads to each library pool.

Note: For example, if a cDNA sample is 100 μL, add (0.8 x 100 μL) = 80 μL of SPRI beads.
SECTION: Section 1: Hybridization
1.9. Vortex the tube(s) for 5 sec. Briefly centrifuge (~2 sec).
SECTION: Section 1: Hybridization
1.10. Incubate tube(s) at room temperature for 5 minutes.
SECTION: Section 1: Hybridization
1.11. Place the tube(s) in an 1.5 mL magnetic rack and wait for all the beads to bind to the magnet (~3 min: liquid should be clear).

Critical! Ensure the supernatant is completely clear before proceeding. Discarding any beads in the supernatant will result in a reduction of transcripts and genes detected per cell.
SECTION: Section 1: Hybridization
1.12. With tube(s) still on the magnetic rack, slowly remove and discard the clear supernatant.
SECTION: Section 1: Hybridization
1.13. Without resuspending beads, add 300 μL of 85% ethanol to each tube using a P1000 and wait 1 minute.
SECTION: Section 1: Hybridization
1.14. Using a pipette, aspirate and discard the ethanol from each tube.
SECTION: Section 1: Hybridization
1.15. Without resuspending beads, add another 300 μL of 85% ethanol to each tube using a P1000 and wait 1 minute.
SECTION: Section 1: Hybridization
1.16. Using a pipette, aspirate and discard all of the ethanol from each tube (it may be necessary to remove the final few drops with a P20 pipette). With the tube still on the rack, air dry the beads (~2 min).

Critical! Do NOT over-dry the beads. Over-drying of beads can lead to substantial losses in yield. “Cracking” of the beads is a sign of over-drying
SECTION: Section 1: Hybridization
1.17. Remove tube(s) from the magnetic rack. Proceed directly to Section 1.2.
2. 1.2 Hybridization
2.1. Add the following reagents individually to each cDNA sample from step 1.1.17 . Mix by flicking
the 1.5 mL tube(s). If making a master mix for multiple samples, scale up volumes by 10%.

 

2.2. For each cDNA sample, spin down and carefully transfer the entire volume (12 μL) into new 0.2 mL tube(s) using a P20 set to 15 μL.
2.3. In a new 0.2 mL tube(s) for each cDNA sample, prepare a Probe Solution as follows. Mix by flicking the tube(s). If making a master mix for multiple samples, scale up volumes by 10%.
 

2.4. Heat the Probe Solution(s) for 2 minutes at 95°C in a thermocycler with the lid heated to 105°C, then immediately cool for 5 minutes on ice.
2.5. Heat the cDNA sample(s) for 5 minutes at 95°C in a thermocycler with the lid heated to 105°C, then immediately place at room temperature.
2.6. Incubate the Probe Solution(s) and cDNA sample(s) at room temperature for 5 minutes.
2.9. Transfer the entire volume (28 μL) of the Probe Solution into each cDNA sample to create the Hybridization Reaction.
2.10. Vortex the tube(s) for 5 sec. Briefly centrifuge (~2 sec).
2.11. Without mixing, add 30 μL of Hybridization Enhancer to the top of the meniscus of the Hybridization Reaction from step 9.

Note: Do not mix after adding the Hybridization Enhancer. It should form a distinct layer above the hybridization reaction.
2.12. Briefly centrifuge tube(s) to ensure there are no bubbles present.
2.13. Ensure tubes are tightly capped to prevent excess evaporation.
2.14. Incubate each Hybridization Reaction at 70°C for 15-17 hours in a thermocycler with the lid at 85°C.

Critical! Rapid transfer directly from the thermocycler at 70°C is a critical step for minimizing off-target binding. Do not remove the Hybridization Reaction tube(s) from the thermocycler or otherwise allow it to cool to less than 70°C until Step 2.1.12.
2.7. Set a thermocycler to 70°C with the lid at 85°C for infinite time.
2.8. Vortex the tube(s) for 5 sec. Briefly centrifuge (~2 sec).
SECTION: Section 2: Capture and Amplification
3. 2.1 Bind and Wash
SECTION: Section 2: Capture and Amplification
3.1. Prepare the following:
Gather two heat blocks, set one to 68°C (for 1.5 mL tube) and the other to 48°C (for 2 mL and 5 mL tubes).
Program a thermocycler to 95°C and set the heated lid to 105°C.
Equilibrate Streptavidin Binder Beads to room temperature for at least 30 minutes.
Equilibrate SPRI beads (Ampure XP or KAPA Pure Beads) to room temperature for at least 30 minutes.
Prepare at least 400 μL of 85% ethanol per reaction.
Take out magnetic rack for 1.5 mL tubes.
SECTION: Section 2: Capture and Amplification
3.2. Note: For performing gene capture on cDNA, you need to use reagents from the WT or WT Mega cDNA Amplification  box: Amplification Reaction Solution and Amplification Master Buffer.

Gather the following items and handle as indicated below:

 

SECTION: Section 2: Capture and Amplification
3.3. Check if precipitate is dissolved, then equilibrate the Binding Buffer to room temperature. Keep Bead Wash Buffer A and Bead Wash Buffer B in their respective heat blocks.
SECTION: Section 2: Capture and Amplification
3.4. Ensure the Streptavidin Binder Beads have been equilibrated to room temperature for at least 30 minutes. Vortex the Streptavidin Binder Beads until mixed.
SECTION: Section 2: Capture and Amplification
3.5. Add 100 μL of Streptavidin Binder Beads to a new 1.5 mL tube for each hybridization reaction.
SECTION: Section 2: Capture and Amplification
3.6. Add 200 μL of Binding Buffer to the tube with Streptavidin Binder Beads. Mix thoroughly by pipetting up and down 10x with a P200 set to 200 μL.
SECTION: Section 2: Capture and Amplification
3.7. Place the tube(s) in an 1.5 mL magnetic rack and wait for all the beads to bind to the magnet (~2 min: liquid should be clear).
SECTION: Section 2: Capture and Amplification
3.8. Using a pipette, aspirate and discard the supernatant from each tube. Remove tube(s) from the magnetic rack.
SECTION: Section 2: Capture and Amplification
3.9. Repeat steps 6-8 twice for a total of three washes
SECTION: Section 2: Capture and Amplification
3.10. Add 200 μL of Binding Buffer and resuspend the beads by vortexing until fully homogenized.
SECTION: Section 2: Capture and Amplification
3.11. Heat the resuspended beads for 68°C for 10 minutes before proceeding to the next step.
SECTION: Section 2: Capture and Amplification
3.12. Set a P200 pipette to 80 μL. After the hybridization reaction from Section 1.2 is complete, open the thermocycler lid and immediately transfer the entire volume of each Hybridization Reaction into a corresponding tube of preheated Streptavidin Binder Beads from step 11. Mix by pipetting and flicking.

Critical! Rapid transfer directly from the thermocycler at 70°C is a critical step for minimizing off-target binding. Do not remove the Hybridization Reaction tube(s) from the thermocycler or otherwise allow it to cool to less than 70°C before transferring the solution to the washed Streptavidin Binding Beads. Allowing the Hybridization Reaction to cool to room temperature for more than 5 minutes will result in as much as 10–20% increase in off-target binding.
SECTION: Section 2: Capture and Amplification
3.13. Incubate the tube(s) containing the Hybridization Reaction and Streptavidin Binder Beads mixture at 68°C for 5 minutes.
Critical! Do not vortex. Aggressive mixing is not required.
SECTION: Section 2: Capture and Amplification
3.14. Remove the tube(s) from the heat block. Briefly centrifuge (~2 sec).
SECTION: Section 2: Capture and Amplification
3.15. Place the tube(s) in a magnetic rack and wait for all the beads to bind to the magnet (~2 min: liquid should be clear).
SECTION: Section 2: Capture and Amplification
3.16. Using a pipette, aspirate and discard the clear supernatant from each tube.
Note: Some Hybridization Enhancer reagent residue may be visible after supernatant removal and throughout each wash step. This will not affect the final capture product.
SECTION: Section 2: Capture and Amplification
3.17. Remove the tube(s) from the magnetic rack and add 200 μL of 68°C Bead Wash Buffer A. Mix by pipetting.
SECTION: Section 2: Capture and Amplification
3.18. Incubate the tube(s) at 68°C for 5 minutes and place Bead Wash Buffer A back into the 68°C heat block.
SECTION: Section 2: Capture and Amplification
3.19. Briefly centrifuge the tube(s).
SECTION: Section 2: Capture and Amplification
3.20. For each hybridization reaction, transfer the entire volume from step 19 (~200 μL) into new 1.5 mL tube(s).
Critical! This step reduces background from non-specific binding to the surface of the tube.
SECTION: Section 2: Capture and Amplification
3.21. Place the tube(s) in an 1.5 mL magnetic rack and wait for all the beads to bind to the magnet (~1 min: liquid should be clear).
SECTION: Section 2: Capture and Amplification
3.22. Using a pipette, aspirate and discard the clear supernatant from each tube.
SECTION: Section 2: Capture and Amplification
3.23. Remove the tube(s) from the magnetic rack and add 200 μL of 48°C Wash Buffer B. Mix by pipetting and briefly centrifuge.
SECTION: Section 2: Capture and Amplification
3.24. Incubate the tube(s) at 48°C for 5 minutes and place Bead Wash Buffer B back into the 48°C heat block.
SECTION: Section 2: Capture and Amplification
3.25. Place the tube(s) in a magnetic rack and wait for all the beads to bind to the magnet (~1 min: liquid should be clear).
SECTION: Section 2: Capture and Amplification
3.26. Using a pipette, aspirate and discard the clear supernatant from each tube.
SECTION: Section 2: Capture and Amplification
3.27. Repeat steps 23-26 twice for a total of three washes.
SECTION: Section 2: Capture and Amplification
3.29. Remove any residual supernatant with a P20 pipette. Proceed immediately to the next step.
SECTION: Section 2: Capture and Amplification
3.30. New Step 30: Remove the tube(s) from the magnetic rack and resuspend in 100 μL of the Amplification Reaction Solution. Mix by pipetting until homogenized, move to PCR strip tubes, then incubate on ice. This solution will be referred to as Streptavidin Binding Bead Slurry. 
Note: Move slurry to PCR tubes for the following amplification reaction.
SECTION: Section 2: Capture and Amplification
4. 2.2 PCR and SPRI Clean Up
SECTION: Section 2: Capture and Amplification
4.1. Start cDNA amplification. 
Note: For primer annealing, steps 3 and 6 below (*) have different time and temperature settings. Double check the settings you input into the thermocycler before starting the amplification protocol.

 

 

Note: No 2nd cycling!
SECTION: Section 2: Capture and Amplification
4.2. As soon as the program reaches 4°C, place the tube(s) at room temperature and proceed immediately to the next step.
SECTION: Section 2: Capture and Amplification
4.3. Ensure the SPRI beads have been equilibrated to room temperature for at least 30 minutes. Vortex the SPRI beads until fully mixed. Add 80 μL of SPRI beads (0.8x) to each tube. Mix by vortexing for 5 sec.
Note: It is not necessary to recover supernatant or remove Streptavidin Binding Beads from the amplified PCR product.
SECTION: Section 2: Capture and Amplification
4.4. Incubate tube(s) at room temperature for 5 minutes
SECTION: Section 2: Capture and Amplification
4.5. Place the tube(s) on the high position of the magnetic rack for 0.2 mL tubes and wait for all the beads to bind to the magnet (~2 min: liquid should be clear).
SECTION: Section 2: Capture and Amplification
4.6. With tubes still on the magnetic rack, remove and discard the clear supernatant.
SECTION: Section 2: Capture and Amplification
4.7. With tubes still on the magnetic rack, add 180 μL of 85% ethanol to each tube.
SECTION: Section 2: Capture and Amplification
4.8. Incubate tube(s) at room temperature for 1 minute.
SECTION: Section 2: Capture and Amplification
4.9. With tubes still on the magnetic rack, remove and discard the supernatant.
SECTION: Section 2: Capture and Amplification
4.10. Repeat steps 10-12 once more for a total of 2 washes. Remove any residual ethanol with a P20 pipette.
SECTION: Section 2: Capture and Amplification
4.11. With tubes still on the magnetic rack, air dry the beads (~30 sec). 
Critical! Do NOT over-dry the beads. Over-drying of beads can lead to substantial losses in yield. “Cracking” of the beads is a sign of over-drying.
SECTION: Section 2: Capture and Amplification
4.12. Remove the tube(s) from the magnetic rack. Fully resuspend each bead pellet with 32 μL of nuclease-free water.
SECTION: Section 2: Capture and Amplification
3.28. While incubating the tubes at 48°C during the wash steps, in a new 1.5 mL tube, make the Amplification Reaction Solution by adding Amplification Master Buffer and Amplification Primer Mix. Mix well and store on ice. 

For example, if processing one cDNA sample, add 60.5 μL of Amplification Primer Mix to 60.5 μL of Amplification Master Buffer.


 

SECTION: Section 2: Capture and Amplification
4.13. Incubate the tube(s) at room temperature for 5 minutes.
SECTION: Section 2: Capture and Amplification
4.14. Place the tube(s) on the low magnet position of the magnetic rack for 0.2 mL tubes and wait for all the beads to bind to the magnet (~2 min: liquid should be clear).
SECTION: Section 2: Capture and Amplification
4.15. With tubes still on the magnetic rack, transfer 30 μL of the supernatant containing the purified enriched cDNA into new 0.2 mL tube(s). Store on ice.
SECTION: Section 2: Capture and Amplification
5. 2.3 Enriched cDNA Quantification
SECTION: Section 2: Capture and Amplification
5.1. Measure the concentration of the cDNA using the Qubit dsDNA HS protocol.
Note: Be sure to record sample concentrations as they will be needed for further downstream steps.
SECTION: Section 2: Capture and Amplification
5.2. Run 1 µL of the cDNA on a Bioanalyzer or TapeStation. Use the concentration obtained from the Qubit to determine the appropriate dilution necessary (check manufacturer specifications, 1:10 dilution is generally appropriate). 


SECTION: Section 2: Capture and Amplification
5.3. Captured cDNA can be stored at this point at 4C for up to 2 days or at -20C for up to 3 months. If you wish to continue, proceed directly to 

1. Short-read protocol: Section 3: Preparing Libraries for Sequencing in either the Evercode WT or Evercode WT Mega protocol.

2. Long-read protocol: ONT Library Prep for Split-seq cDNA.

[STOPPING POINT].