TITLE: R2C2 protocol draft
AUTHORS: Alison Tang

[DESCRIPTION]
Total RNA is reverse transcribed and PCR amplified using the smartseq2 system (with indexed oligo-dTs). Complementary DNA is then size selected for transcripts >=2.5 kb with a low melt agarose gel extraction. Size-selected cDNA and non-size-selected cDNA are pooled. Finally, long concatemers are generated from the cDNA with the R2C2 protocol. The resulting R2C2 cDNA can be sequenced on the nanopore following Oxford Nanopore Technology's genomic DNA library preparation protocol.

[STEPS]
SECTION: cDNA synthesis, adding oligo-dT indexes
7. Add 6 µL mix #2 to each tube (10 µL total volume).

SECTION: cDNA synthesis, adding oligo-dT indexes
1. Thaw oligo-dT, dNTP, DTT, Superase-In, 5x SmartScribe buffer, TSO SmartSeq primer on ice.
SECTION: cDNA synthesis, adding oligo-dT indexes
3. Make mix #2,  6 µL for each reaction.
 

SECTION: cDNA synthesis, adding oligo-dT indexes
8. Incubate tubes of RNA+Mix#2 in thermocycler:
42 °C, 90 min
70 °C, 5 min
4 °C,    

SECTION: cDNA synthesis, adding oligo-dT indexes
2. If you are size selecting, make four mixes of Mix #1 with four different oligo-dT indexes.
Otherwise, you will only need as many tubes of Mix #1 as you have samples that will be pooled together. So for 2 samples to be pooled together, you will need 2 tubes each with 2 ul of Mix #1 with two different oligo-dT indexes.
 




SECTION: cDNA synthesis, adding oligo-dT indexes
5. Add 2 µL of diluted RNA to mix #1 with one oligo-dT index. If size selecting, then add the remaining 2 µLto another aliquot of mix #1 with a different oligo-dT index.  Repeat for each sample.
 


SECTION: cDNA synthesis, adding oligo-dT indexes
6. Incubate tubes of Mix#1+RNA at 72 °C for 3 min. Prepare to snap cool after.

SECTION: cDNA synthesis, adding oligo-dT indexes
4. Thaw the total RNA samples. Dilute each sample such that you have 4 µL if size selecting, or 2 µL if not, of 0.5 µg/µL total RNA.

SECTION: cDNA synthesis, adding oligo-dT indexes
6.1. Snap cool on ice after denaturing to prevent secondary structure from reforming. Then pre-warm the thermocycler to 42 °C for RT reaction.

SECTION: cDNA synthesis, adding oligo-dT indexes
2.1. Aliquot 2 µL from the tubes of mix #1 into each PCR tube (only necessary if you'll have multiple pools to be sequenced).

SECTION: cDNA amplification
9. Thaw ISPCR primers and KAPA Hifi HotStart on ice.
SECTION: cDNA amplification
10. Make amplification mix (15 µLeach reaction).

 

SECTION: cDNA amplification
11. Add 15 µLamplification mix to each cDNA synthesis reaction (25 µL total volume).

SECTION: cDNA amplification
12. Incubate tubes in thermocycler:
37 °C, 10 min
95 °C, 3 min

followed by 12 (or however many) cycles of:
98 °C, 20 s
67 °C, 15 s
72 °C, 390 s

then,
72 °C, 10 min final extension
4 °C,   


SECTION: Bead cleanup
13. Add 20 µL of Ampure XP beads (0.8:1 bead ratio) to enough low-bind tubes as you have PCR reactions.
 


SECTION: Size selection (cDNA, gel extraction)
19. Set up a 1% low melt agarose gel, ~100 ul TAE for a ~12 well gel with wide wells. 
 

SECTION: Size selection (cDNA, gel extraction)
20. Load:
1 well -- diluted 1kb ladder, 10 ul, + 6X loading dye containing Sybr Gold
Other well(s) -- sample(s), ~30 ul, + 6X loading dye containing Sybr Gold

SECTION: Bead cleanup
16. Elute in 20 µLof water. A smaller elution volume can be more effective for pooling the samples. Resuspend and then incubate for 10 min at 37 °C. Then place on magnetic rack and remove eluate into new tubes.

SECTION: Size selection (cDNA, gel extraction)
21. Run at 80V until separation around 2.5 kb is easier to see.
SECTION: Size selection (cDNA, gel extraction)
22. Transfer gel to blue light box. Excise 2.5 kb+ and place gel slice into a 2 ml tube.

 

SECTION: Size selection (cDNA, gel extraction)
23. Weigh gel slices, using an empty 2 ml tube to tare. 
 

SECTION: Size selection (cDNA, gel extraction)
24.  Add β-agarase buffer to each tube. The μl of buffer to add is 2 volume's worth, or twice the number of grams of gel slice. 

SECTION: Size selection (cDNA, gel extraction)
25. Incubate aton ice for20 min.

 

SECTION: Size selection (cDNA, gel extraction)
26. Remove buffer, then repeat steps 24 and 25.
SECTION: Size selection (cDNA, gel extraction)
27. Remove buffer, then incubate gel slices at 65 °C for10 min.

SECTION: Size selection (cDNA, gel extraction)
28. Transfer tubes to 42 °C heat block. Wait 1 minute for the melted gel to cool down to avoid inactivating beta-agarase in the next step.

 

SECTION: Size selection (cDNA, gel extraction)
26.1. Set up a heat block at 65 °C and another heat block at 42 °C.

SECTION: Size selection (cDNA, gel extraction)
29. Add 2 ul of beta-agarase per 300 g of gel.

 

SECTION: Bead cleanup
14. Transfer the amplified product into the low bind tubes with beads. Pipette up and down 10 times, spin down, and then incubate at 42 Room temperaturefor5 min.

SECTION: Bead cleanup
15. Transfer tubes to a magnetic rack. Pipette off liquid. Wash the beads twice in 70% EtOH without disturbing the bead pellet.
 

SECTION: Bead cleanup
17. Qubit the purified cDNA (dsDNA HS).
SECTION: Bead cleanup
18. Pool one control and one experimental sample together s.t. the concentration of the samples are equal. If you aren't size selecting, you can skip the size selection section. Else, pool the other ctrl and expt samples that contain the other two indexes. Make note of which two oligo-dT indexes will be size-selected. 
SECTION: Size selection (cDNA, gel extraction)
30. Incubate at 42 °C for 60 min, or overnight.

 

SECTION: Size selection (cDNA, gel extraction)
31. Place tubes on ice for 5 min.

SECTION: Size selection (cDNA, gel extraction)
32. Centrifuge at 15000 x g for 7 minto pellet any undigested gel.

SECTION: Size selection (cDNA, gel extraction)
33. Remove the supernatant and place into a low-bind tube.
SECTION: Size selection (cDNA, gel extraction)
34. Add 0.7:1 beads and follow the bead purification protocol above. Incubate sample and beads on a hula mixer. Elute in30 µLof water. Exact volume isn't too important here.

 

SECTION: Size selection (cDNA, gel extraction)
35. Qubit (dsDNA HS).

SECTION: Circularization
37. Combine 200 ng of splint with 200 ng of cDNA. Water can be used to adjust the volume to 20 µL if necessary.
 

 


SECTION: Circularization
38. Incubate at 50 °Cfor 60 min.

SECTION: Size selection (cDNA, gel extraction)
36. Combine size-selected ctrl+expt cDNA with non-size-selected ctrl+expt cDNA from step 18 s.t. the concentrations of all four oligo-dT indexes are comparable. 
SECTION: Circularization
39. Prepare a mastermix of exonucleases, 30 µL per reaction.



 



SECTION: Circularization
41. Incubate at 37 °C  60 min, or for at least 360 min.

SECTION: Circularization
43. Add40 µLof beads (0.8:1) to each reaction. Follow with bead cleanup protocol and elute in 30 µL H2O for three rolling circle amplification reactions, else elute in 10*(the number of RCA reactions desired).

 

SECTION: Rolling circle amplification
44. Make phi29 mastermix.

 

SECTION: Circularization
40. Add 30 ul of exonuclease mastermix to each reaction.
SECTION: Rolling circle amplification
45. Add 10 µL of circularized cDNA to 40 µLof phi29 mastermix.

SECTION: Rolling circle amplification
46. Incubate RCA reactions at 30 °C 360 min.

SECTION: Rolling circle amplification
47. Add 2 µL T7 endonuclease to each reaction. Pipette gently since the RCA cDNA is long.

SECTION: Rolling circle amplification
48. Incubate for 120 min at37 °C. Agitate occasionally. 
 


SECTION: Size selection (RCA cDNA, gel extraction)
52. Set up a 1% low melt agarose gel, ~100 ul TE for a ~12 well gel with wide wells.
SECTION: Rolling circle amplification
50. Clean the sheared combined RCA cDNA with the Zymo DNA Clean and Concentrator-5 kit. Use 2:1 binding buffer, change collection tubes to do an extra 1 minute dry spin, and elute in 20 µL. Exact volume isn't too important here.

SECTION: Rolling circle amplification
51. Dilute samples 1:10 and qubit (dsDNA HS).
SECTION: Rolling circle amplification
49. Assemble a ~26 gauge needle with a syringe. Pull up the 3+ replicates of RCA T7 reactions into the syringe, combining them. Expel the reactions. Repeat the in+out four more times, for five times total. Three is ok too.
SECTION: Size selection (RCA cDNA, gel extraction)
53. Load:
1+ wells -- diluted 1:50 NEB 1kb ladder, 10 ul, + 6X loading dye containing Sybr Gold
? wells -- sample + 6X loading dye containing Sybr Gold

 

SECTION: Size selection (RCA cDNA, gel extraction)
54. Follow steps for running the gel, gel excision, buffer exchange, agarose digestion, bead cleanup (all detailed in the size selection section). You don't need to run the gel for as long since you only need to be able to cut out the 4+ kb molecules. Elute in 50 µL (nanopore SQK-LSK110 library prep takes 48 ul max, 1+ ug of cDNA).

SECTION: Circularization
42. Incubate at 80 °C for 20 min to deactivate.