TSS-MPRA Protocol
Carlos Guzman, Sascha Duttke, Camila De Arruda Saldanha, Christopher Benner, Sven Heinz
Abstract
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
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Make sure that cells are resuspended in media/PBS before TRIzol LS extraction (250 uL)
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Wipe down work surface and pipettes with RNAse Zap
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Cool down centrifuge to 4℃
Steps
RNA Extraction
Add 750µL of 250µL 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).
Incubate at RT for 0h 5m 0s.
Add 230µL of 0h 0m 15s.
Incubate for 0h 5m 0s at RT.
Centrifuge samples for 0h 15m 0s (13,000 x g, 4℃).
Transfer the aqueous layer containing the RNA to a new 1.5 mL LoBind tube (~ 400 - 500 uL).
Add 1µL of gly
Add 1/10th volume of 3M NaOAc (5.5) to each sample.
Vortex 0h 0m 5s and add 1x volume of isopropanol to each sample. Mix by inverting 10 times and then spin down briefly.
Incubate ON at -20°C (optionally 20 minutes at -20℃).
Centrifuge samples for 0h 30m 0s (MAX x g, 4℃).
Discard supernatant, wash with 1mL 75% EtOH. Discard EtOH, quick-spin, remove rest of EtOH.
Air dry pellet at RT until it is translucent (~ 0h 3m 0s). ( Optional: pellet can be frozen at -80°C indefinitely)
Resuspend in 30µL TE’T [0.05% volume Tween-20, 0.1millimolar (mM) EDTA, 10millimolar (mM) Tris pH 7.5].
Capped MPRA 5' RNA-seq
Aliquot 15µL of sample into 1.5 mL epi-tube and incubate at 75°C for 0h 2m 0s, then chill on ice for 0h 2m 0s
Add 35µL of CIP1 master-mix [25.25µL of ddH2O + 0.05% Tween-20, 5µL0.75µL 0.5µL 2µL 37°C for 1h 0m 0s.
Incubate at 75°C for 0h 1m 0s, then chill on ice for 0h 2m 0s
Add 10µL CIP 2 master-mix [8.5µL of ddH2O + 0.05% Tween-20, 1µL0.5µL 37°C for 0h 30m 0s.
Add 500µL 0h 5m 0s.
Add 140µL of TE’T and 140µL of
Vortex well, then centrifuge samples for 0h 10m 0s at 12,000 x g (RT).
Transfer the upper layer into new tubes.
Add 1µL of gly
Add 1/10th volume of 3M NaOAc (5.5) to each sample.
Vortex 0h 0m 5s and add 1x volume of isopropanol to each sample. Mix by inverting 10 times and then spin down briefly.
Incubate 0h 10m 0s at -20°C (optionally 20 minutes at -20℃).
Centrifuge samples for 0h 30m 0s (MAX x g, 4℃).
Discard supernatant, wash with 1mL 75% EtOH. Discard EtOH, quick-spin, remove rest of EtOH.
Air dry pellet at RT until it is translucent (~ 0h 3m 0s). ( Optional: pellet can be frozen at -80°C indefinitely)
Resuspend RNA in 6µL TET [0.05% volume Tween-20, 1millimolar (mM) EDTA, 10millimolar (mM) Tris pH 7.5].
Denature RNA at 75°C for 0h 2m 0s, then chill on ice for 0h 2m 0s.
Add 9µL of RppH master-mix to each sample, mix really well and incubate at 37°C for 1h 0m 0s
Add 10µL 5’ Ligation master-mix, mix well and incubate at 21°C for 2h 0m 0s or 16°C for 16h 0m 0s.
Resuspend pellet in 7µL of Annealing master-mix.
Denature RNA at 75°C for 0h 2m 0s, then incubate at 56°Cfor 0h 45m 0s, then cool on ice.
Add 13µL RT master-mix, incubate at 50°C for 1h 0m 0s.
Do PCR
| A | B | C |
|---|---|---|
| PCR Program | ||
| 94C | 30 seconds | |
| 15x cycles | 94C | 15 seconds |
| 63C | 30 seconds | |
| 70C | 18 seconds*** | |
| 70C | 5 minutes | |
| 4C | hold infinity |
the extension time depends on the length of your inserts! LongAmp copies DNA at 1kb per 50 seconds
Add 1µL 0h 15m 0s at 37°C.
Add 47µL 20% PEG / 2.5M NaCl + 3µL µL. Wash 2x with 80% EtOH. Air-dry beads until cracked (~12 minutes). Resuspend in 15µL of 1x 0h 5m 0s.
Run the entire sample on 0.5µL of any 25bp ladder (80V for 0h 15m 0s, then 180V for 1h 15m 0s ). Perforate 0.5 mL qubit tubes 4x with 22g needle, and place qubit tubes inside 1.5 mL LoBind tube.
Stain the gel with 20mL of 1x TBE. Buffer + 3µL of 0h 5m 0s inside a RNase free container in the dark.
Visualize gel and cut gel depending on desired size (adapters are ~118 bp, add to size you expect).
Centrifuge samples for 0h 5m 0s (20,000 x g , RT). Ensure that all the gel is crushed and spun down into
Add 160µL of TET to each.
Shake samples for 0h 45m 0s at RT (do not shake too quickly).
Transfer slurry to 0h 2m 0s at 1000 x g.
Add 100 µL of TET + 300 mM NaCl to column and incubate for 0h 30m 0s at RT, then spin for 0h 2m 0s at 1000 x g.
Clean up in 10µL EB.
