TITLE: Modified protocol for genome-wide mapping of uncapped transcripts (GMUCT) in eukaryotes
AUTHORS: Diep R Ganguly, Garima Bhatia, Susheel Sagar Bhat, Brian D Gregory

[DESCRIPTION]
This is a modified protocol to perform genome-wide mapping of uncapped and cleaved transcripts (GMUCT; Willmann et al, 2014 Methods, 10.1016/j.ymeth.2013.07.003).

[BEFORE_START]
Purify DNA-free total RNA samples using method of choice, for example, TRIzol-chloroform extraction (dx.doi.org/10.17504/protocols.io.bt8wnrxe).

[GUIDELINES]
Make sure you are working at a clean work bench with filter tips to minimize chances for any sample contamination, especially RNases. Keep all samples and enzyme mixes on ice in between steps.

[STEPS]
SECTION: Poly-A selection
1. For each sample, dilute 5 - 30 μg total RNA in 100 μL of nuclease-free water (or 10 mM Tris-Cl pH 7.5).
SECTION: Poly-A selection
2. Perform poly-A RNA selection using Dynabeads mRNA purification kit as per the manufacturer's instructions (see attachments).

Note, the capacity of these beads are 75 μg total RNA. So, if you are starting with < 32.5 μg total RNA, you may want to scale-down all the reactions by one-half in order to conserve reagents.
SECTION: Ligate 5&#x27; adapter
5. Combine the following in 200 μL PCR tubes, for each sample, and mix by pipetting 6 times. 

 

SECTION: Ligate 5&#x27; adapter
6. Incubate at 70 ºC for 2 min, then 4 ºC for at least 2 min.
SECTION: Ligate 5&#x27; adapter
7. Prepare the following ligation mix, multiplying the volumes for the number of samples being processed (+5% for pipetting errors). Mix with gentle pipetting then briefly centrifuge.

 

SECTION: Ligate 5&#x27; adapter
8. Add 4.5 μL of the mix to each sample, mix by pipetting 6 times, then briefly spin down.
SECTION: Ligate 5&#x27; adapter
9. Incubate at 28 ºC for 1 h.
SECTION: Ligate 5&#x27; adapter
10. Proceed directly to the next section or store at -20 ºC overnight.
SECTION: Poly-A selection
11. Another poly-A selection is performed to remove un-ligated adapter using the NEBNext Poly(A) mRNA Magnetic Isolation Module. This kit is much more cost-effective to perform poly-A selection on samples with <5 μg RNA.
SECTION: Poly-A selection
12. Add 35 μL DEPC-treated water to all samples to bring the final volume to 50 μL.
SECTION: Poly-A selection
13. In a second 0.2 mL PCR tube, aliquot 20 μL of well resuspended NEBNext Magnetic Oligo d(T)25 Beads.
SECTION: Poly-A selection
14. Add 100 μL RNA Binding Buffer and pipette mix 6 times.
SECTION: Poly-A selection
15. Place tubes containing beads on a magnetic rack until solution is clear.
SECTION: Poly-A selection
16. Remove supernatant without disturbing the beads.
SECTION: Poly-A selection
17. Remove tubes from the rack and repeat steps 12 - 14.
SECTION: Poly-A selection
18. Resuspend beads in 50 μL RNA Binding Buffer.    
SECTION: Poly-A selection
19. Add 50 μL of ligated RNA sample and mix by pipetting 6 times.
SECTION: Poly-A selection
20. Incubate at 65 ºC for 5 minutes then hold at 4 ºC (denature RNA and facilitate binding of the poly-A to oligo dT beads).
SECTION: Poly-A selection
21. Remove tubes from thermal cycler when samples reach 4 ºC.
SECTION: Poly-A selection
22. Resuspend beads by pipetting 6 times, then incubate for 5 minutes at room temperature.
SECTION: Poly-A selection
3. Determine concentration and purity of poly-A selected RNA using a Nanodrop.
SECTION: Poly-A selection
4. Aliquot a normalized amount of poly-A selected RNA, for 5' adapter, in 9 μL of DEPC-treated water. Ideally, this would be approximately 600 ng poly-A RNA recovered from 30 μg total RNA.
SECTION: Poly-A selection
23. Repeat step 22.
SECTION: Poly-A selection
24. Place tubes on magnetic rack until solution clear (poly-A RNA bound to beads).
SECTION: Poly-A selection
25. Discard all supernatant without disturbing beads.
SECTION: Poly-A selection
26. Remove tubes from magnetic rack.
SECTION: Poly-A selection
27. Wash beads by adding 200 μL Wash Buffer and pipetting 6 times.
SECTION: Poly-A selection
28. Place tubes on the rack and wait for the solution to become clear.
SECTION: Poly-A selection
29. Discard the supernatant then remove tubes from the rack.
SECTION: Poly-A selection
30. Repeat steps 27 - 29.
SECTION: Poly-A selection
31. Add 8 μL of Tris Buffer and mix by pipetting 6 times.
SECTION: Poly-A selection
32. Incubate samples at 80 ºC for 2 minutes, then hold at 25 ºC.
SECTION: Poly-A selection
33. Once samples reach 25 ºC, place immediately on the magnetic rack.
SECTION: Poly-A selection
34. Once solution is completely clear, transfer the supernatant to a clean nuclease-free PCR Tube. Keep tube on ice if continuing with reverse transcription reaction or store at -20 ºC.
SECTION: Reverse transcription and addition of 3&#x27; adapter
35. Set up the following program in a thermal cycler:
65 °C for 5 min
Hold at 4 °C
25 °C for 10 min
42 °C for 60 min
85 °C for 5 min
Hold at 4 °C

Run the program to allow the thermal cycler to preheat (pause on step 1).
SECTION: Reverse transcription and addition of 3&#x27; adapter
36. Prepare the following in clean 200 μL PCR tubes for each sample

 

SECTION: Reverse transcription and addition of 3&#x27; adapter
37. Mix contents of each tube by pipetting 6 times.
SECTION: Reverse transcription and addition of 3&#x27; adapter
38. Place tubes in pre-heated thermal cycler and run steps 1-2, allows samples to sit at 4 °C for at least 2 minutes.
SECTION: Reverse transcription and addition of 3&#x27; adapter
39. Prepare the following reverse transcription mix. Multiply volumes per sample and add 5% to account for pipetting errors. Mix gently by pipetting, then briefly centrifuge.

 


SECTION: Reverse transcription and addition of 3&#x27; adapter
40. Add 10 μL of the mix to each sample (keep on ice) and mix by pipetting up and down 6 times, then centrifuge briefly.
SECTION: Reverse transcription and addition of 3&#x27; adapter
41. Return samples to the thermal cycler and continue with steps 3-6.
SECTION: PCR amplification
42. Set up the following program in a thermal cycler:
98 °C for 30 sec
98 °C for 10 sec
60 °C for 30 sec
72 °C for 15 sec
Back to step 2 (11x)
72 °C for 10 min
Hold at 4 °C

Run the program to allow the thermal cycler to preheat (pause on step 1).
SECTION: PCR amplification
43. Add 2 μL of a unique RNA PCR Primer Index (RPI#, see materials) to each sample and mix by pipetting 6 times. Use the Illumina Index Adapter Pooling Guide (TruSeq Small RNA Library Prep Kit) to aid with RPI# selection (where # = 1-48).
SECTION: PCR amplification
44. Prepare the following PCR mix, multiplying the volumes for the number of samples (+5 % extra for pipetting errors). Mix thoroughly by pipetting and briefly centrifuge.

 

SECTION: PCR amplification
45. Add 78 μL of the PCR mix to each sample and mix by pipetting up and down 6 times. Centrifuge briefly and keep samples on ice.
SECTION: PCR amplification
46. Distribute 100 μL across 4 PCR tubes (i.e. 4 x 25 μL reactions per sample). This is done to increase the PCR efficiency.
SECTION: PCR amplification
47. Place all samples into the pre-heated thermal cycler and run the program.
SECTION: Ethanol precipitation
48. Combine 25 μL aliquots into a single 1.5 mL microtube.
SECTION: Ethanol precipitation
49. Precipitate DNA by adding 500 μL ethanol, 3.5 μL GlycoBlue, and 10 μL 3 M NaOAc (pH 5.5).
SECTION: Ethanol precipitation
50. Leave samples at -80 °C for at least 2 hours.
SECTION: Ethanol precipitation
51. Centrifuge at max speed for 45 min at 4 °C.
SECTION: Ethanol precipitation
52. Remove supernatant and rinse with 750 μL 80% ethanol.
SECTION: Ethanol precipitation
53. Centrifuge at max speed for 5 min at 4 °C.
SECTION: Ethanol precipitation
54. Remove supernatant without disturbing pellet and allow to air dry for 1-2 min.
SECTION: Ethanol precipitation
55. Resuspend in 10 μL of nuclease-free water and allow samples to resuspend for 20 minutes on ice.
SECTION: Size selection
56. Setup a 6% TBE polyacrylamide gel (Invitrogen) in a gel running tank with 1x TBE buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA pH 8).
SECTION: Size selection
57. Prepare ladder and samples:
- Mix 1.5 μL 25 bp ladder (Invitrogen) with 8.5 μL DEPC water and 10 μL gel loading buffer II (Invitrogen or NEB).
- Mix 10 μL sample with 10 μL gel loading buffer II.
SECTION: Size selection
58. Load ladder and and samples into TBE gel, keeping one lane free between each sample to minimize likelihood of cross-contamination and make gel excision easier.
SECTION: Size selection
59. Run gel at 155 V for approximately 30 minutes (dye front should be just touching the bottom of the gel).

Note, you may prefer to run the gel at 80-100 V for 1-1.5 hours to achieve better separation of adapter-adapter clones (see below). You could also use a higher percentage of polyacrylamide.
SECTION: Size selection
60. While the gel is running, prepare gel breaker tubes for each sample to be size selected (or nuclease-free 0.5 mL tubes).
SECTION: Size selection
61. Use a 21G needle to make holes in the bottom of the tubes (or, in the case of gel breaker tubes, to increase their size) and place within a nuclease free 2 mL microtube.
SECTION: Size selection
62. Once gel run has completed, incubate gel for 10 minutes with 1X GelStar Nucleic Acid Stain in 100-200 mL 1X TBE in a nuclease-free tray.
SECTION: Size selection
63. Rinse gel with distilled water 3 times then image on a UV transilluminator.
SECTION: Size selection
64. For each sample, excise the region corresponding to 135 - 500 bp (can include larger fragments if there is visible product) and place inside gel breaker tubes (nested in a 2 mL microtube). Keep the excised region consistent among samples within an experiment.

Note: a minimum size of 135 bp is recommended since this corresponds to a 17 bp insert and anything smaller becomes much more difficult to map uniquely to the genome. This also avoids adapter-adapter clones (produced from random hexamer binding to 5' adapter), the longest of which would be 118 bp.
SECTION: Size selection
65. Centrifuge at max speed for 2 minutes and ensure that the gel completely passes through the holes (repeat spin and add more holes if necessary).
SECTION: Size selection
66. Add 300 μL NEB Buffer 2 (50 mM NaCl, 10 mM Tris-Cl, 10 mM MgCl2. 1 mM DTT, pH 7.9) and rotate for at least 2 hours (or overnight at 4 ºC).
SECTION: Size selection
67. Precipitate RNA by adding 5 μL GlycoBlue, 30 μL 3 M NaOAc pH 5.5, and 900 μL ethanol.
SECTION: Size selection
68. Incubate at -80 ºC for at least 2 hours (can leave overnight to maximize recovery).
SECTION: Size selection
69. Centrifuge at max speed for 1 hour at 4 ºC.
SECTION: Size selection
70. Remove supernatant and wash with 750 μL 70% ethanol.
SECTION: Size selection
71. Centrifuge at max speed for 5 min at 4 ºC.
SECTION: Size selection
72. Remove as much supernatant as possible, and allow tubes to air dry for 1 -2 minutes.
SECTION: Size selection
73. Quantify library concentration using a Qubit fluorometer with Qubit dsDNA HS Assay Kit (Invitrogen). Alternatively, run libraries on a LabChip GXII or Bioanalyzer to give information on concentration and fragment sizes.
SECTION: Size selection
64.1.