TITLE: Brad-seq mRNA (for Shotgun or DGE)
AUTHORS: Yin-Tse  Huang

[DESCRIPTION]
Brad-seq mRNA

[STEPS]
SECTION: Tissue Lysis
1. Add 5 µL of 2-Mercaptoethanol (2-ME) to 1 mL of LBB (for ratio, adjust it for the amount LBB used)


SECTION: Tissue Lysis
2. Wipe off RNAlatter from tissue; Place 20 mgof tissue in crushing tube with metal cone
 
SECTION: Tissue Lysis
3. Add 200 µL in tube
SECTION: Tissue Lysis
4. Crush sample with multi-beads shocker at 2000 rpm 
SECTION: Tissue Lysis
5. Sit in Room temperature for 5 min  and remove metal cone

SECTION: Tissue Lysis
6. Centrifuge at 14000 rpm, 1 min  

SECTION: Tissue Lysis
7. Transfer all the lysate to a new 1.5 mL tube

SECTION: Tissue Lysis
8. Centrifuge at 14000 rpm, 10 min 

SECTION: Tissue Lysis
9. Carefully transfer the supernatant to a new tube. Be careful not to carry over cell debris

 

SECTION: 1st mRNA extraction
10. Put 100 µL lysed sample in each well of 8-strip (Keep another half in -80 °C just in case)

SECTION: 1st mRNA extraction
11. Add 2 µL of biotin-20nt-20T oligo; Mix well by pipetting

SECTION: 1st mRNA extraction
12. Sit in Room temperature for 10 min for incubation
SECTION: 1st mRNA extraction
12.1. While waiting, prepare NEB magnetic Streptavidin beads
Resuspend beads well before use
SECTION: 1st mRNA extraction
12.2. Dispense 20 µL of Streptavidin beads into each well of a 8-strip (1 mg:1 µL beads/tissue ratio); Put 8-strip on magnetic rack and remove supernatant
SECTION: 1st mRNA extraction
12.3. Resuspend beads with 100 µL LBB to wash the beads;
Place 8-strip on magnet rack and remove supernatant
Beads are ready for use
SECTION: 1st mRNA extraction
13. Add biotin-incubated samples to washed beads
SECTION: 1st mRNA extraction
14. Slowly stir at Room temperature  at 500 rpm for 10 min   
SECTION: 1st mRNA extraction
15. Place 8-strip on magnet rack and remove supernatant
 

 

SECTION: 1st mRNA extraction
16. Wash with 150 µL of cold WBA (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant
SECTION: 1st mRNA extraction
17. Wash with 150 µL of cold WBB (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant

SECTION: 1st mRNA extraction
18. Wash with 150 µL of cold LSB (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant
SECTION: 1st mRNA extraction
19. RNA elution buffer prep.
 


SECTION: 1st mRNA extraction
20. Resuspend beads in 17 µL RNA elution buffer
SECTION: 1st mRNA extraction
21. Warm at 80 °C for 2 min  in a thermal cycler. After that, cool it quickly on ice for 5 min  
 

SECTION: 1st mRNA extraction
22. Place 8-strip on magnet rack and transfer supernatant (17 µL ) to a new 8-strip
 


SECTION: 1st mRNA extraction
22.1. DNAase treatment for Secondary mRNA Recovery (for SHO protocol)
SECTION: 1st mRNA extraction
22.2.   
DANase prep.
 

SECTION: 1st mRNA extraction
22.3. Add 2.31 µL DNAase to 1st RNA supernatant (17 µL ) = 19.31 µL in total


SECTION: 1st mRNA extraction
22.4. Sit at Room temperature for 15 min 
SECTION: 1st mRNA extraction
22.5. Kill the activity of DNAase at 70 °C for 10 min 

SECTION: 2nd mRNA recovery
23. Add 150 µL DEPC water to re-suspend the used beads; 
Place 8-strip on magnet rack and remove supernatant

SECTION: 2nd mRNA recovery
24. Add 5 µL of 2-Mercaptoethanol (2-ME) to 1 mL of SBB (for ratio, adjust it for the amount SBB used)
SECTION: 2nd mRNA recovery
25. Add 150 µL SBB and Place 8-strip on magnet rack and remove supernatant (Wash beads)

SECTION: 2nd mRNA recovery
26. Add in DNAase treated RNA (19.31 µL ) to the washed beads + 130 µL SBB

SECTION: 2nd mRNA recovery
27. Sit at Room temperature for 10 min 

SECTION: 2nd mRNA recovery
28. Place 8-strip on magnet rack and remove supernatant
SECTION: 2nd mRNA recovery
29. Wash with 150 µL of cold WBA (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant

SECTION: 2nd mRNA recovery
30. Wash with 150 µL of cold WBB (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant

SECTION: 2nd mRNA recovery
31. Wash with 150 µL of cold LSB (keep it as cool as possible) and Place 8-strip on magnet rack and remove supernatant
SECTION: 2nd mRNA recovery
32. Resuspend beads in 17 µL RNA elution buffer
SECTION: 2nd mRNA recovery
33. Warm at 80 °C for 2 min  in a thermal cycler. After that, cool it quickly on ice for 5 min  
 

SECTION: 2nd mRNA recovery
34. Place 8-strip on magnet rack and transfer supernatant (17 µL ) to a new 8-strip
 

 

SECTION: RNA fragmentation &amp; 3-prime adapter cDNA priming
35. Make 3 strand priming 2.5 µL 

 

SECTION: RNA fragmentation &amp; 3-prime adapter cDNA priming
36. Mix 2.5 µL3 strand priming with 7.5 µL extracted RNA =  10 µL in total
Keep the remaining at-20 °C )

SECTION: RNA fragmentation &amp; 3-prime adapter cDNA priming
37. 10 µL of mixed in thermo cycler for RNA fragmentation
 

 

SECTION: RNA fragmentation &amp; 3-prime adapter cDNA priming
38. 5 µL master mix + 10 µL fragmented RNA = 15 µL mixed solution
1st strand master mix
 

SECTION: RNA fragmentation &amp; 3-prime adapter cDNA priming
39. Mixed solution in thermo cycler for reverse transcription
 


SECTION: RNA fragmentation &amp; 3-prime adapter cDNA priming
40. Use Ampure beads solution for cDNA purification;
35 µL Ampure bead solution + 20 µL cDNA
Ampure bead solution
 

SECTION: RNA fragmentation &amp; 3-prime adapter cDNA priming
41. Vortex for 5 min at Room temperature 

SECTION: RNA fragmentation &amp; 3-prime adapter cDNA priming
42. Place 8-strip on magnet rack and remove supernatant
SECTION: RNA fragmentation &amp; 3-prime adapter cDNA priming
43. Wash with 200 µL ethanol without suspending; Dry the pellet
Repeat this process twice 

 


 

SECTION: 5-prime adapter sequence addition
44. Add 4 µL of 10 μM 5-prime adapter directly to the pellet atRoom temperature  to resuspend the bead pellet

SECTION: 5-prime adapter sequence addition
45. Prepare the master mix in advance during cDNA synthesis, and add the enzyme just before using
6 µL mater mix/rxn + 4 µL suspended cDNA pellet
mater mix recipe
 

SECTION: 5-prime adapter sequence addition
46. Sit at Room temperature for 15 min 

SECTION: 5-prime adapter sequence addition
47. 40 µL Ampure bead solution + 10 µL cDNA 
Ampure bead solution recipe
 

SECTION: 5-prime adapter sequence addition
48. Vortex for 5 min at Room temperature 

SECTION: 5-prime adapter sequence addition
49. Place 8-strip on magnet rack and remove supernatant
SECTION: 5-prime adapter sequence addition
50. Wash with 200 µL ethanol without suspending; Dry the pellet
Repeat this process twice 

 


SECTION: 5-prime adapter sequence addition
51. Elute the pellet in 20 µL of 10 mM Tris pH 8.0; 
Let it sit for 1 min

 

SECTION: 5-prime adapter sequence addition
52. Transfer the supernatant to new tubes
SECTION: Enrichment and adapter extension
53. 12.2 µL Enrichment master mix + 2 µL 1 μM ILL-INDEX primer +  5.8 µL cDNA
Enrichment master mix recipe
 

SECTION: Enrichment and adapter extension
54. Mixed solution in thermo cycler

 

SECTION: Final Cleanup
55. 24 µLAmpure beads + 20 µLof enrichment product; 
Mix well

SECTION: Final Cleanup
56. Vortex for 5 min at Room temperature 

SECTION: Final Cleanup
57. Place 8-strip on magnet rack and remove supernatant
SECTION: Final Cleanup
58. Wash with 200 µL ethanol without suspending; Dry the pellet
Repeat this process twice 

 


SECTION: Final Cleanup
59. Elute the pellet in 12 µL of H2O

SECTION: Final Cleanup
60. Use Bioanalyzer for quantification for measuring the concentration of each sample
SECTION: Library preparation
61. Mix equimolar of samples in a 1.5 mL tube
SECTION: Library preparation
62. Add same amount of AMpure XP as the DNA, mix well, and let it stand for 5 min

SECTION: Library preparation
63. Place on magnet rack for 5 min, remove supernatant and wash twice with 70 µL 80% EtOH. Dry.

SECTION: Library preparation
64. Resuspend the beads by adding 22 µL of water, leave them in the magnet rack for 5 min 
Transfer the supernatant to a new 0.5 mL low bind tube.
SECTION: Library preparation
65. Quantification with Qbit or Bioanalyzer