TITLE: R2C2 protocol using 10x Single-Cell RNA-seq Assay cDNA
AUTHORS: Xian Adiconis, Joshua Z Levin

[DESCRIPTION]
This protocol is based on the published Rolling Circle to Concatemeric Consensus (R2C2) method (1). The aim is to make elongated cDNA via rolling circle amplification using cDNA generated from the 10x Chromium 3' (or 5') single-cell RNA-seq assay to increase the accuracy of downstream sequencing on an Oxford Nanopore Technologies sequencing platform. The major modification from the original protocol is to add an additional cDNA re-amplification step (see section "10x cDNA re-amplification") to enrich for fragments with the proper sequence (poly(T) at 3' and TSO at 5') at both ends, which is essential in circularization with the 10x Splint fragment.

[STEPS]
SECTION: Splint generation
1.  Prepare the following on ice
 
 
 
SECTION: Splint generation
2. Run the following program
Thermocycler Conditions: (Lid@105 °C, 50 µl)
 
 95 °C, 3 min
followed by 1 cycle of:
98 °C, 1 min
62 °C, 1 min
72 °C, 6 min
then
4 °C,   

SECTION: Splint generation
3. Cleanup with Select-a-Size columns (Zymo) and a size cut-off of ~125 bp. Follow the Select-a-Size cleanup protocol for single-size selection, but in the buffer preparation add 85 µL of 100% ethanol to 500 µL of Select-a-Size DNA binding buffer. Elute in 15 µL DNA elution buffer (from the kit). QC with 1/5x dilution for Quant-it (Thermo Fisher Scientific) and 1/50x dilution for BioAnalyzer DNA HS assay (Agilent).
Label this product as "10x Splint", store at -20 °C for later use.

SECTION: 10x cDNA re-amplification
4. Enrichment PCR
Prepare the follow reaction mix on ice
 
 
 
SECTION: 10x cDNA re-amplification
5. Run the following program
Thermocycler Conditions: (Lid@105 °C, 100 µl)
 
 98 °C, 45 s
followed by 10 cycle of:
98 °C, 10 s
62 °C, 15 s
72 °C, 3 min
then
72 °C, 5 min 
4 °C,   
SECTION: cDNA circularization
7. Circularization

Prepare the following mix
 
 
Incubate @50 °C  for 60 min 
Then add the following mix
 
 
 Incubate @37 °C for 360 min , then @80 °C for 20 min 
Cleanup the reaction with 40 µL SPRI beads (0.8x)

SECTION: cDNA circularization
8. Circularization product purification
SECTION: cDNA circularization
8.1. Add 40 µL(0.8x) of resuspended, room-temperature AMPure XP SPRI beads to the reaction mix. Pipette mix 10 times. Perform a quick spin to collect all liquid from the sides of the tube.

SECTION: cDNA circularization
8.2. Incubate sample on bench top for 5 min at Room temperature.
SECTION: cDNA circularization
8.3. Place the tube on a magnetic stand to separate the beads from the supernatant. Use a P200 pipettor to remove the supernatant.
SECTION: cDNA circularization
8.4. Wash 2 times with 200 µL of freshly prepared 80% ethanol. After removal of the second wash of 200 µL of ethanol, spin the tube strip briefly, return to magnetic stand and remove residual ethanol with a P20 pipettor. Do not let the beads dry out.

SECTION: cDNA circularization
8.5. Remove the tube from the magnetic stand. Immediately add 30 µL of H2O and pipette mix to resuspend. Perform a quick spin to collect all liquid from the sides of the tube. Place at Room temperaturefor 5 min to elute the cDNA from the beads.
SECTION: 10x cDNA re-amplification
6.4. Wash 2 times with 200 µL of freshly prepared 80% ethanol. After removal of the second wash of 200 µL of ethanol, spin the tube strip briefly, return to magnetic stand and remove residual ethanol with a P20 pipettor. Do not let the beads dry out.
SECTION: 10x cDNA re-amplification
6.3. Place the tube on a magnetic stand to separate the beads from the supernatant. Use a P200 pipettor to remove the supernatant.
SECTION: 10x cDNA re-amplification
6.2. Incubate sample on bench top for 5 min at Room temperature.
SECTION: 10x cDNA re-amplification
6.1. Add 95 µL of resuspended, room-temperature ProNex beads to the PCR mix. Pipette mix 10 times. Perform a quick spin to collect all liquid from the sides of the tube.
SECTION: 10x cDNA re-amplification
6. cDNA purification
SECTION: 10x cDNA re-amplification
6.5. Remove the tube from the magnetic stand. Immediately add 15 µL of buffer EB (10 mM Tris-HCl, pH 8.5) and pipette mix to resuspend. Perform a quick spin to collect all liquid from the sides of the tube. Place at Room temperaturefor 5 min to elute the DNA from the beads.
SECTION: 10x cDNA re-amplification
6.6. Place the tube on a magnetic stand to separate the beads from the supernatant. Transfer the eluted cDNA samples to a new tube.
SECTION: 10x cDNA re-amplification
6.7. QC with 1/5x dilution for Quant-it and BioAnalyzer DNA HS assay. Store the remaining product at -20 °C for later use.
SECTION: cDNA circularization
8.6. Place the tube on a magnetic stand to separate the beads from the supernatant. Transfer the eluted cDNA samples to a new tube.
SECTION: Rolling circle amplification
9. Prepare the following mix, then split into 3 reaction wells of 50 µL each.
 
 

Incubate in a thermocycler  @30 °C for   

SECTION: Rolling circle amplification
10. SPRI clean and T7 endonuclease treatment
Pool all reactions and adjust volume to 300 µL with H2O. Add150 µL150 µL SPRI beads (0.5x) to your DNA and leave on a rotator for 5 minutes to mix. If not homogeneous, flick the tube to mix.
Notes: After adding the AMPure XP SPRI beads and mixing by inverting the tubes, there's uneven distribution of the beads, mostly the sign of longer cDNA strand.
Place on a magnet stand until a complete pellet forms, wash twice with 500 µL 70% ethanol. After the ethanol removal, add the following mix to re-suspend the beads.
 
 
Incubate on a thermal shaker at 37 °C at 1000 RPM for 120 min. Agitate the tube occasionally to help the solution homogenize.
Place on a magnet stand to pellet the beads. Recover the supernatant that contains the DNA to a new tube. Purify the DNA with 0.5x volume of AMPure XP SPRI beads, wash twice with 500 µL 70% ethanol. Elute in 20 µL H2O.
QC with Quant-it for concentration, run BioAnalyzer DNA 12000 assay (Agilent) or 1% agarose gel to visualize the size distribution. The majority of the fragments should be > 10kb.


 

 
The elongated cDNA is now ready for downstream Oxford Nanopore Technologies (ONT) library prep.
SECTION: Rolling circle amplification
9.1. From this point on, be really careful with the DNA since it will shear pretty easily. Do not pipet up and down and try not to vortex.