TITLE: FLAM-seq (with Kapa mRNA enrichment)_CMS_edit-2024-09-25
AUTHORS: Corneliu Sologon

[DESCRIPTION]
fork__CMS_edit-2024-09-25

[STEPS]
SECTION: GI Tailing (using USB poly(A) tail length assay, Thermo Fisher).
18. Prepare on ice (20 uL total):
14 µL 
4 µL 
2 µL 
SECTION: GI Tailing (using USB poly(A) tail length assay, Thermo Fisher).
20. 1.5 µL 
4 °C 2 min 
SECTION: GI Tailing (using USB poly(A) tail length assay, Thermo Fisher).
21. 38.7 µL 
4 Room temperature 5 min 
SECTION: GI Tailing (using USB poly(A) tail length assay, Thermo Fisher).
22. 20 Room temperature 3 min 
Discard supernatant
SECTION: GI Tailing (using USB poly(A) tail length assay, Thermo Fisher).
23. 50 µL 30 s 
Discard supernatant
SECTION: GI Tailing (using USB poly(A) tail length assay, Thermo Fisher).
25. 10 s 
Discard supernatant
SECTION: GI Tailing (using USB poly(A) tail length assay, Thermo Fisher).
26. Remove from magnet
17 µL 
  

SECTION: cDNA synthesis (using SMARTscribe reverse transcriptase kit, Clontech).
30.   
10 s 
SECTION: cDNA synthesis (using SMARTscribe reverse transcriptase kit, Clontech).
29. In 0.2 mL PCR tube from step 27
16 µL  
2 µL  
SECTION: cDNA synthesis (using SMARTscribe reverse transcriptase kit, Clontech).
28. Prepare RT master mix at room temperature (22 uL total; 1.1X per sample):
8 µL 
1.5 µL 
4 µL 
2 µL 
2 µL 
2 µL 
2.5 µL 
SECTION: cDNA synthesis (using SMARTscribe reverse transcriptase kit, Clontech).
32. 24 µL 
4 Room temperature 5 min 
SECTION: cDNA library amplification (using Advantage 2 PCR enzyme system, Clontech).
39. Prepare the cDNA library amplification rxn:
40 µL 
10 µL 
2 µL 
2 µL
2 µL
42 µL 
2 µL 
SECTION: GI Tailing (using USB poly(A) tail length assay, Thermo Fisher).
19. 37 °C 60 min 
SECTION: GI Tailing (using USB poly(A) tail length assay, Thermo Fisher).
24.   

SECTION: GI Tailing (using USB poly(A) tail length assay, Thermo Fisher).
27. 16 µL   to new 0.2 mL PCR tube
SECTION: cDNA synthesis (using SMARTscribe reverse transcriptase kit, Clontech).
31. 72 °C 3 min 
42 °C HOLD - add 22 µL 
42 °C 60 min 
70 °C 10 min 
4 °C HOLD

SECTION: cDNA synthesis (using SMARTscribe reverse transcriptase kit, Clontech).
33. 20 Room temperature 3 min 
Discard supernatant
SECTION: cDNA synthesis (using SMARTscribe reverse transcriptase kit, Clontech).
34. 50 µL 30 s 
Discard supernatant
SECTION: cDNA synthesis (using SMARTscribe reverse transcriptase kit, Clontech).
35.   

SECTION: cDNA synthesis (using SMARTscribe reverse transcriptase kit, Clontech).
36. 10 s 
Discard supernatant
SECTION: cDNA synthesis (using SMARTscribe reverse transcriptase kit, Clontech).
37. Remove from magnet
42 µL 
  

SECTION: cDNA synthesis (using SMARTscribe reverse transcriptase kit, Clontech).
38. 40 µL   to new 0.2 mL PCR tube
SECTION: Sequencing library preparation (using the SMRTbell™ Template Prep Kit)
48. Performed in PacBio sequencing core
SECTION: cDNA library amplification (using Advantage 2 PCR enzyme system, Clontech).
47. 40 µL   to new 0.2 mL PCR tube
SECTION: cDNA library amplification (using Advantage 2 PCR enzyme system, Clontech).
46. Remove from magnet
42 µL 
  

SECTION: cDNA library amplification (using Advantage 2 PCR enzyme system, Clontech).
45. 10 s 
Discard supernatant
SECTION: cDNA library amplification (using Advantage 2 PCR enzyme system, Clontech).
44.   

SECTION: cDNA library amplification (using Advantage 2 PCR enzyme system, Clontech).
43. 200 µL 30 s 
Discard supernatant
SECTION: cDNA library amplification (using Advantage 2 PCR enzyme system, Clontech).
42. 20 Room temperature 3 min 
Discard supernatant
SECTION: cDNA library amplification (using Advantage 2 PCR enzyme system, Clontech).
41. 60 µL 
4 Room temperature 5 min 
SECTION: cDNA library amplification (using Advantage 2 PCR enzyme system, Clontech).
40. Perform PCR
1. 98 °C HOLD - add cDNA rxn
2.98 °C 1 min 
3.98 °C 10 s 
4.63 °C 15 s 
5.68 °C 3 min repeat 40.3 -40.5 24X
6.68 °C 7 min 
7.4 °C HOLD
SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
17. 16 µL   to new 0.2 mL PCR tube

SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
16. 20 Room temperature 5 min 
SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
15. 70 °C 2 min 

SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
14. Remove from magnet
16 µL 
  
SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
13. 20 Room temperature 5 min 
Discard supernatant
SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
12. Remove from magnet
200 µL 
  
SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
11. 20 Room temperature 5 min
Discard supernatant
SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
10. 50 µL 
  
20 °C 5 min 
SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
9. 70 °C 2 min 
20 °C 5 min 
10 s 
SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
8. 50 µL
  
SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
7. 20 Room temperature 5 min
Discard supernatant
SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
6. Remove from magnet
200 µL
  

SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
5. 20 Room temperature 5 min 
Discard supernatant

SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
4. 65 °C 2 min 
20 °C 5 min 
10 s 

SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
3. 50 µL 
  

SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
2. 2-10 µg   in 0.2 mL PCR tube
50 µL
SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
1. Prepare mRNA Beads
SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
1.1. 52.5 µL can be scaled for multiple samples
  

SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
1.2. 20 Room temperature 5 min 
Discard supernatant
SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
1.3. Remove from magnet
52.5 µL 
  
SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
1.4. 20 Room temperature 5 min 
Discard supernatant
SECTION: Poly(A)+ RNA preparation (using Kapa mRNA Beads)
1.5. Remove from magnet
52.5 µL