TITLE: TSS-MPRA Protocol
AUTHORS: Carlos Guzman, Sascha Duttke, Camila De Arruda Saldanha, Christopher Benner, Sven Heinz

[DESCRIPTION]
Cis-regulatory elements can be classified by the shapes of their transcription initiation patterns, which are indicative of distinct regulatory mechanisms. While massively parallel reporter assays (MPRAs) have enabled the functional study of sequence features within regulatory elements on an unprecedented scale, current MPRA approaches focus on quantifying transcript abundance, largely ignoring where transcription starts. This information however, could provide evidence that regulatory mechanisms in the context of the reporter assay resemble those active in the genome. Here we describe a transcription start site-capturing massively parallel reporter assay (TSS-MPRA) that simultaneously measures the location and frequency of transcription initiation. We characterize the degree to which plasmid-based MPRAs recapitulate endogenous initiation patterns (“TSS shapes”) and transcription levels and evaluate the effects of increasing insert length and reporter chromatinization on plasmid-derived transcription initiation. Employing a new bioinformatic approach to compare TSS shapes, we find that shorter, episomal constructs most faithfully replicate endogenous initiation patterns and transcription levels. Finally, we illustrate how TSS-MPRA can be used to decode cis-regulatory grammar by assessing the effects of core promoter and transcription factor motif mutations and single nucleotide polymorphisms on transcription initiation. Taken together, TSS-MPRA reveals important caveats to consider when using MPRAs and enables high-resolution analysis of the sequence grammar underlying transcription initiation.  

[BEFORE_START]
* Make sure that cells are resuspended in media/PBS before TRIzol LS extraction (250 uL)
* Wipe down work surface and pipettes with RNAse Zap
* Cool down centrifuge to 4℃

[STEPS]
SECTION: RNA Extraction
1. Add 750 µL of   per 250 µL of sample volume (3x) and pipette up and down 5x (Optional: samples can be stored at 4 °C ON or at -20 °Cfor up to a year).

SECTION: RNA Extraction
2. Incubate at RT for 5 min.

SECTION: RNA Extraction
3. Add 230 µL of   and shake vigorously by hand for 15 s.

SECTION: RNA Extraction
4. Incubate for 5 min at RT.

SECTION: RNA Extraction
5. Centrifuge samples for 15 min (13,000 x g, 4℃).

SECTION: RNA Extraction
6. Transfer the aqueous layer containing the RNA to a new 1.5 mL LoBind tube (~ 400 - 500 uL).

SECTION: RNA Extraction
7. Add 1 µL of gly   to each sample.

SECTION: RNA Extraction
8. Add 1/10th volume of 3M NaOAc (pH 5.5) to each sample.

SECTION: RNA Extraction
9. Vortex 5 s and add 1x volume of isopropanol to each sample. Mix by inverting 10 times and then spin down briefly.

SECTION: RNA Extraction
10. Incubate ON at -20 °C (optionally 20 minutes at -20℃).

SECTION: RNA Extraction
11. Centrifuge samples for 30 min (MAX x g, 4℃).
SECTION: RNA Extraction
12. Discard supernatant, wash with 1 mL 75% EtOH. Discard EtOH, quick-spin, remove rest of EtOH.
SECTION: RNA Extraction
13. Air dry pellet at RT until it is translucent (~ 3 min). (Optional: pellet can be frozen at -80 °C indefinitely)

SECTION: RNA Extraction
14. Resuspend in 30 µL TE’T [0.05 % volume Tween-20, 0.1 millimolar (mM) EDTA, 10 millimolar (mM) Tris pH 7.5].

SECTION: Capped MPRA 5&#x27; RNA-seq
15. Aliquot 15 µL of sample into 1.5 mL epi-tube and incubate at 75 °C for 2 min, then chill on ice for 2 min

SECTION: Capped MPRA 5&#x27; RNA-seq
16. Add 35 µL of CIP1 master-mix [25.25 µL of ddH2O + 0.05% Tween-20, 5 µL , 0.75 µL   , 0.5 µL  , 2 µL   ] to each sample and incubate at 37 °C for 60 min.


SECTION: Capped MPRA 5&#x27; RNA-seq
17. Incubate at 75 °C for 1 min, then chill on ice for 2 min

SECTION: Capped MPRA 5&#x27; RNA-seq
18. Add 10 µL CIP 2 master-mix [8.5 µL of ddH2O + 0.05% Tween-20, 1 µL , 0.5 µL  ] to each sample and incubate at 37 °C for 30 min.

SECTION: Capped MPRA 5&#x27; RNA-seq
19. Add 500 µL  , vortex, and incubate at RT for 5 min.

SECTION: Capped MPRA 5&#x27; RNA-seq
20. Add 140 µL of TE’T and 140 µL of to each sample.

SECTION: Capped MPRA 5&#x27; RNA-seq
21. Vortex well, then centrifuge samples for 10 min at 12,000 x g (RT).

SECTION: Capped MPRA 5&#x27; RNA-seq
22. Transfer the upper layer into new tubes.
SECTION: Capped MPRA 5&#x27; RNA-seq
23. Add 1 µL of gly to each sample.
SECTION: Capped MPRA 5&#x27; RNA-seq
24. Add 1/10th volume of 3M NaOAc (pH 5.5) to each sample.
SECTION: Capped MPRA 5&#x27; RNA-seq
25. Vortex 5 s and add 1x volume of isopropanol to each sample. Mix by inverting 10 times and then spin down briefly.
SECTION: Capped MPRA 5&#x27; RNA-seq
26. Incubate 10 min at -20 °C (optionally 20 minutes at -20℃).
SECTION: Capped MPRA 5&#x27; RNA-seq
27. Centrifuge samples for 30 min (MAX x g, 4℃).
SECTION: Capped MPRA 5&#x27; RNA-seq
28. Discard supernatant, wash with 1 mL 75% EtOH. Discard EtOH, quick-spin, remove rest of EtOH.
SECTION: Capped MPRA 5&#x27; RNA-seq
29. Air dry pellet at RT until it is translucent (~ 3 min). (Optional: pellet can be frozen at -80 °C indefinitely)
SECTION: Capped MPRA 5&#x27; RNA-seq
30. Resuspend RNA in 6 µL TET [0.05 % volume Tween-20, 1 millimolar (mM) EDTA, 10 millimolar (mM) Tris pH 7.5].

SECTION: Capped MPRA 5&#x27; RNA-seq
31. Denature RNA at 75 °C for 2 min, then chill on ice for 2 min.

SECTION: Capped MPRA 5&#x27; RNA-seq
32. Add 9 µL of RppH master-mix to each sample, mix really well and incubate at 37 °C for 60 min

 

 

SECTION: Capped MPRA 5&#x27; RNA-seq
33. Add 10 µL 5’ Ligation master-mix, mix well and incubate at 21 °C for 120 min or 16 °C for 960 min.
 

 


SECTION: Capped MPRA 5&#x27; RNA-seq
34.   

SECTION: Capped MPRA 5&#x27; RNA-seq
35.   

SECTION: Capped MPRA 5&#x27; RNA-seq
36.   

SECTION: Capped MPRA 5&#x27; RNA-seq
37.   

SECTION: Capped MPRA 5&#x27; RNA-seq
38.   

SECTION: Capped MPRA 5&#x27; RNA-seq
39.   

SECTION: Capped MPRA 5&#x27; RNA-seq
40.   

SECTION: Capped MPRA 5&#x27; RNA-seq
41.   

SECTION: Capped MPRA 5&#x27; RNA-seq
42.   

SECTION: Capped MPRA 5&#x27; RNA-seq
43.   

SECTION: Capped MPRA 5&#x27; RNA-seq
44.   

SECTION: Capped MPRA 5&#x27; RNA-seq
45. Resuspend pellet in 7 µL of Annealing master-mix.

 

SECTION: Capped MPRA 5&#x27; RNA-seq
46.  Denature RNA at 75 °C for 2 min, then incubate at 56 °Cfor 45 min, then cool on ice.

SECTION: Capped MPRA 5&#x27; RNA-seq
47. Add 13 µL RT master-mix, incubate at 50 °C for 60 min.

 

SECTION: Capped MPRA 5&#x27; RNA-seq
48. Do PCR

 

 

 

SECTION: Capped MPRA 5&#x27; RNA-seq
49. Add 1 µL  , incubate for 15 min at 37 °C.

SECTION: Capped MPRA 5&#x27; RNA-seq
50. Add 47 µL 20% PEG / 2.5M NaCl + 3 µL   to each sample. Incubate at RT for  . Wash 2x with 80% EtOH. Air-dry beads until cracked (~12 minutes). Resuspend in 15 µL of 1x  . Incubate at RT for 5 min.

SECTION: Capped MPRA 5&#x27; RNA-seq
51. Run the entire sample on   with 0.5 µL of any 25bp ladder (80V for 15 min, then 180V for 75 min ). Perforate 0.5 mL qubit tubes 4x with 22g needle, and place qubit tubes inside 1.5 mL LoBind tube.

SECTION: Capped MPRA 5&#x27; RNA-seq
52. Stain the gel with 20 mL of 1x TBE. Buffer + 3 µL of   for 5 min inside a RNase free container in the dark.

SECTION: Capped MPRA 5&#x27; RNA-seq
53. Visualize gel and cut gel depending on desired size (adapters are ~118 bp, add to size you expect).

 

SECTION: Capped MPRA 5&#x27; RNA-seq
54. Centrifuge samples for 5 min (20,000 x g , RT). Ensure that all the gel is crushed and spun down into  .

SECTION: Capped MPRA 5&#x27; RNA-seq
55. Add 160 µL of TET to each.

SECTION: Capped MPRA 5&#x27; RNA-seq
56. Shake samples for 45 min at RT (do not shake too quickly).

SECTION: Capped MPRA 5&#x27; RNA-seq
57. Transfer slurry to  and spin for 2 min at 1000 x g.

SECTION: Capped MPRA 5&#x27; RNA-seq
58. Add 100 µL of TET + 300 mM NaCl to column and incubate for 30 min at RT, then spin for 2 min at 1000 x g.

SECTION: Capped MPRA 5&#x27; RNA-seq
59. Clean up in   columns according to instructions. Elute in 10 µL EB.