TITLE: Rapid Ribosome (Polysome) Profiling
AUTHORS: Jason D Limberis, Yuxiang Chen

[DESCRIPTION]
Ribosome profiling is a powerful technique used to study translation at a genome-wide level. It involves the sequencing of ribosome-protected mRNA fragments to determine the positions of ribosomes on transcripts. This information can be used to infer translation rates and identify translated open reading frames. While traditional ribosome profiling methods can be time-consuming and expensive, our method is rapid and cost-effective, and the inclusion of UMIs allows for precise quantification.

[BEFORE_START]
Read through the protocol and ensure you have the correct reagents depending on your chosen approach

[STEPS]
SECTION: Linker ligation
21. Add 1 µL of 100 micromolar (µM) RiboS_linker (linker RNA oligo) to 10 µL of RNA, denature it for1 min at 70 °C, and then cool it to Room temperature
SECTION: Linker ligation
22. Set up the ligation reaction below and incubate for 90 min at 37 °C
 

SECTION: RT (TSO) reaction
30. 
 
Mix thoroughly by gently pipetting up and down at least 10 times, then centrifuge briefly to collect the solution to the bottom of the tube
Denature for5 min at 70 °C in a thermal cycler and then place on ice for ≥1 min
SECTION: RT (TSO) reaction
31. Vortex the Template Switching RT Buffer briefly followed by a quick spin
Combine the following components in a reaction tube
 
Mix thoroughly by gently pipetting up and down at least 10 times, then centrifuge briefly to collect the solution to the bottom of the tube
SECTION: RT (TSO) reaction
32. Combine 7 µL RT reaction mix (above) with 13 µL of the annealed mix, mix well by gently pipetting up and down at least 10 times, then centrifuge briefly to collect the solution to the bottom of the tube
Incubate as below
 

SECTION: Index PCR
33. 
     Component      50ul        10 µM   Forward Index Primer      2.5        10 µM   Reverse Index Primer      2.5        RiboS_blocking_oligo      2        2X   Phusion Master Mix      25        Template   DNA      18   

 

Fragment is 150bp excluding insert at this stage
AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNGATGGG[INSERT]CAAGCAGAAGACGGCATACGAGAT[i7]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC
SECTION: Index PCR
34. Add 90 µL AMPure XP beads to the PCR product
Incubate at Room temperature for 5 min
Place on a magnetic rack
Aspirate supernatant
Add 200 µL 70 % (v/v) ethanol
Wait for 30 s
Aspirate and discard the supernatant
Add 200 µL 70 % (v/v) ethanol
Wait for 30 s
Aspirate and discard the supernatant
Resuspend beads in 10 µL of H20 
Incubate for 2 min
Transfer to a clean PCR tube
SECTION: Optional: preselection of small RNA fragments
2. Optional but recommended preselection of small RNA fragments using either a urea PAGE gel or bead and membrane selection. 
SECTION: RNA isolation and nuclease footprinting
1. Perform RNA isolation and nuclease footprinting as appropriate for your cell type
(see steps 1-17 here for cultured mammalian cells)
SECTION: Optional: Agarose gel size selection
35. Run a 1% agarose gel, excise the desired band, and perform a gel cleanup.
SECTION: Optional: Depletion of Abundant Sequences by Hybridization
36. To perform DASH to deplete unwanted sequences (e.g., rRNA) follow the protocol here.
SECTION: Quantification
37. Run a tapestation or bioanalyzer chip
Do qubit or qPCR using an Illumina library quant kit to quantify the library and pool the samples (see Illumina Sequencing Coverage Calculator for pooling information)
SECTION: Optional: preselection of small RNA fragments
3. 10 µL RNA plus 30 µL RNA XP beads plus 10 µL 100% isopropanol

SECTION: Optional: preselection of small RNA fragments
10. Transfer the excised gel slice to a 1.5 mL microcentrifuge tube and weigh it.
SECTION: Optional: preselection of small RNA fragments
11. Add 4 volumes of Monarch RNA Cleanup Binding Buffer to the tube with the slice
SECTION: Optional: preselection of small RNA fragments
12. Incubate the sample between 55 °C, gently mixing periodically until the gel slice is completely dissolved (generally <10 min)

SECTION: Optional: preselection of small RNA fragments
13. Add two volumes of absolute ethanol to the sample and mix well by pipetting up and down
SECTION: Optional: preselection of small RNA fragments
14. Incubate the sample between 55 °C or an additional 5 min

SECTION: Optional: preselection of small RNA fragments
15. Insert an RNA cleanup column into a collection tube, load the sample onto the column and close the cap
SECTION: Optional: preselection of small RNA fragments
16. Re-insert the column into the collection tube
SECTION: Optional: preselection of small RNA fragments
17. Transfer the column to a clean 1.5 ml microfuge tube
SECTION: Optional: preselection of small RNA fragments
18. Add 10 µL nucleae-free water directly to the membrane and incubate at Room temperature for 5 min

SECTION: Optional: preselection of small RNA fragments
19. Spin for 1 min
SECTION: Optional: preselection of small RNA fragments
15.1. Spin for 1 min, then discard flow-through
SECTION: Optional: preselection of small RNA fragments
16.1. Add 500 µL RNA Cleanup Wash Buffer

SECTION: Optional: preselection of small RNA fragments
16.2. Spin for 1 min, then discard flow-through
SECTION: Optional: preselection of small RNA fragments
16.3.    for a total of two washes.

SECTION: Optional: preselection of small RNA fragments
3. Add the appropriate volume of 2X RNA loading buffer to each RNA sample
SECTION: Optional: preselection of small RNA fragments
4. Denature the samples for 80 °C 1 min followed by ≥2 min on on ice
SECTION: Optional: preselection of small RNA fragments
5. Load the samples on the polyacrylamide gel with TBE running buffer
SECTION: Optional: preselection of small RNA fragments
7. Separate by electrophoresis for 90 min at 100V
SECTION: Optional: preselection of small RNA fragments
8. Stain the gel for 3 min with 1X SYBR Gold in 1X TBE running buffer on a shaker
SECTION: Optional: preselection of small RNA fragments
9. Visualize the gel and excise desired region (20-40nts)
SECTION: RNA cleanup
23. Add 100 µL RNA Cleanup Binding Buffer

SECTION: Overview
20.   

SECTION: Optional: preselection of small RNA fragments
4. Add 100 µL RNA Cleanup Binding Buffer

SECTION: Optional: preselection of small RNA fragments
6. Insert an RNA cleanup column into a collection tube, load the sample onto the column and close the cap
SECTION: Optional: preselection of small RNA fragments
6.1. Spin for 1 min, then discard flow-through
SECTION: Optional: preselection of small RNA fragments
7. Re-insert the column into the collection tube
SECTION: Optional: preselection of small RNA fragments
7.1. Add 500 µL RNA Cleanup Wash Buffer

SECTION: Optional: preselection of small RNA fragments
7.2. Spin for 1 min, then discard flow-through
SECTION: Optional: preselection of small RNA fragments
7.3.    for a total of two washes.

SECTION: Optional: preselection of small RNA fragments
8. Transfer the column to a clean 1.5 ml microfuge tube
SECTION: Optional: preselection of small RNA fragments
9. Add 10 µL nucleae-free water directly to the membrane and incubate at Room temperature for 5 min

SECTION: Optional: preselection of small RNA fragments
10. Spin for 1 min
SECTION: Optional: preselection of small RNA fragments
3.1. Incubate at Room temperature for 5 min
Place on a magnetic rack
Transfer supernatant to a clean 1.5ml tube
 
SECTION: RNA cleanup
24. Add 150 µL absolute ethanol and mix by pipetting

SECTION: RNA cleanup
25. Insert an RNA cleanup column into a collection tube, load the sample onto the column and close the cap
SECTION: RNA cleanup
25.1. Spin for 1 min, then discard flow-through
SECTION: RNA cleanup
26. Re-insert the column into the collection tube
SECTION: RNA cleanup
26.1. Add 500 µL RNA Cleanup Wash Buffer

SECTION: RNA cleanup
26.2. Spin for 1 min, then discard flow-through
SECTION: RNA cleanup
26.3.    for a total of two washes.

SECTION: RNA cleanup
27. Transfer the column to a clean 1.5 ml microfuge tube
SECTION: RNA cleanup
28. Add 10 µL nucleae-free water directly to the membrane and incubate at Room temperature for 5 min

SECTION: RNA cleanup
29. Spin for 1 min
SECTION: Optional: preselection of small RNA fragments
5. Add 300 µL absolute ethanol and mix by pipetting

SECTION: Overview
11.   

SECTION: Linker ligation
12. Add 1 µL of 100 micromolar (µM) RiboS_linker (linker RNA oligo) to 10 µL of RNA, denature it for1 min at 70 °C, and then cool it to Room temperature
SECTION: Linker ligation
13. Set up the ligation reaction below and incubate for 90 min at 37 °C
 

SECTION: RNA cleanup
14. Add 100 µL RNA Cleanup Binding Buffer

SECTION: RNA cleanup
15. Add 150 µL absolute ethanol and mix by pipetting

SECTION: RNA cleanup
16. Insert an RNA cleanup column into a collection tube, load the sample onto the column and close the cap
SECTION: RNA cleanup
16.1. Spin for 1 min, then discard flow-through
SECTION: RNA cleanup
17. Re-insert the column into the collection tube
SECTION: RNA cleanup
17.1. Add 500 µL RNA Cleanup Wash Buffer

SECTION: RNA cleanup
17.2. Spin for 1 min, then discard flow-through
SECTION: RNA cleanup
17.3.    for a total of two washes.

SECTION: RNA cleanup
18. Transfer the column to a clean 1.5 ml microfuge tube
SECTION: RNA cleanup
19. Add 10 µL nucleae-free water directly to the membrane and incubate at Room temperature for 5 min

SECTION: RNA cleanup
20. Spin for 1 min
SECTION: RT (TSO) reaction
21. 
 
Mix thoroughly by gently pipetting up and down at least 10 times, then centrifuge briefly to collect the solution to the bottom of the tube
Denature for5 min at 70 °C in a thermal cycler and then place on ice for ≥1 min
SECTION: RT (TSO) reaction
22. Vortex the Template Switching RT Buffer briefly followed by a quick spin
Combine the following components in a reaction tube
 
Mix thoroughly by gently pipetting up and down at least 10 times, then centrifuge briefly to collect the solution to the bottom of the tube
SECTION: RT (TSO) reaction
23. Combine 7 µL RT reaction mix (above) with 13 µL of the annealed mix, mix well by gently pipetting up and down at least 10 times, then centrifuge briefly to collect the solution to the bottom of the tube
Incubate as below
 

SECTION: Index PCR
24. 
     Component      50ul        10 µM   Forward Index Primer      2.5        10 µM   Reverse Index Primer      2.5        RiboS_blocking_oligo      2        2X   Phusion Master Mix      25        Template   DNA      18   

 

Fragment is 150bp excluding insert at this stage 
AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNGATGGG[INSERT]CAAGCAGAAGACGGCATACGAGAT[i7]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC
SECTION: Index PCR
25. Add 90 µL AMPure XP beads to the PCR product
Incubate at Room temperature for 5 min
Place on a magnetic rack
Aspirate supernatant
Add 200 µL 70 % (v/v) ethanol
Wait for 30 s
Aspirate and discard the supernatant
Add 200 µL 70 % (v/v) ethanol
Wait for 30 s
Aspirate and discard the supernatant
Resuspend beads in 10 µL of H20 
Incubate for 2 min
Transfer to a clean PCR tube
SECTION: Optional: Agarose gel size selection
26. Run a 1% agarose gel, excise the desired band, and perform a gel cleanup.
SECTION: Optional: Depletion of Abundant Sequences by Hybridization
27. To perform DASH to deplete unwanted sequences (e.g., rRNA) follow the protocol here.
SECTION: Quantification
28. Run a tapestation or bioanalyzer chip
Do qubit or qPCR using an Illumina library quant kit to quantify the library and pool the samples (see Illumina Sequencing Coverage Calculator for pooling information)
SECTION: Analysis
38. Analysis can be done using common pipelines with the addition of umi_tools to count unique reads
6. Pre-run the gel in 1X TBE buffer at 100V for 10min
SECTION: Optional: preselection of small RNA fragments
10.1. Pro Tip: Smashing the gel in the tube increases RNA recovery.