TITLE: Preparation of Encoding Probes SOP005.v1.2                                                (PCR, In-vitro Transcription, Reverse Transcription and USER ENZYME Digest)
AUTHORS: Rory  Kruithoff, Douglas  Shepherd

[DESCRIPTION]
Document Summary:This document, Preparation of Encoding Probes (SOP005), describes the procedure used to produce the final encoding probes used in multiplexed iterative FISH experiments, from commercially-derived, low-yield yet affordable oligo libraries. To prepare the ordered oligo pool into the final encoding probe set, oligos are amplified using limited-cycle PCR, then amplified again and shortened during in-vitro transcription. We follow the amplification steps with reverse transcription to convert our product back into the intended DNA-based, mRNA target complements and then digest away RNA using Uracil-Specific Excision Reagent (USER) enzyme digest to shorten the probes and alkaline hydrolysis to remove RNA nucleotides leaving single-stranded DNA-based encoding probes. Column purification is used to clean the products at various intermediate steps of the protocol and the final product is concentrated using ethanol precipitation prior to use in experiments.   This protocol is strongly derived from Moffitt 2016 (https://doi.org/10.1016/bs.mie.2016.03.020) with some modifications with credit for a majority of this protocol due to Moffitt et al 2016.

[BEFORE_START]
Quick Overview:

Part 1 - PCR Amplification
Step 1 -Prepare the PCR reaction
Step 2- Amplify the template
Step 3 -Purify the template
Step 4–Quality control for template reaction (optional)

Part 2 - In-vitro Transcription
Step 1 – In-vitro Transcription
Step 2 – Quality control of in-vitro transcription (optional)

Part 3 - Reverse Transcription of RNA to DNA
Step 1 – Reverse transcription of mRNA with Uracil Modified Primer.

Part 4 – USER Enzyme Digest
Step 1 – USER Enzyme Digest
Step 2 – Alkaline Hydrolysis
Step 3 – Purification of ssDNA product.
Step 4 – Concentration of Probe.
Step 5 - Quality control of USER enzyme digest product (optional)

v1.2 revision notes
Updated document summary.
Removed primer selection from part 1 which is more applicable to probe design than preparation.

[STEPS]
SECTION: Part 1 - PCR Amplification - Step 1: Prepare the PCR reaction
1. In a 1.7 mL Eppendorf tube, mix the following:
-  40 µL ;
-  2 µL ;
-  2 µL ; 
-  1 µL ; 
-  355 µL ; 
-  400 µL .

SECTION: Part 1 - PCR Amplification - Step 1: Prepare the PCR reaction
2. Aliquot 50 µL  into 16 PCR tubes.
SECTION: Part 1 - PCR Amplification - Step 2: Amplify the template
3. Run the following protocol on a qPCR machine: 
1) 98 °C for 3 min; 
2) 98 °C for 10 s; 
3) *63 °C for 10 s; 
4) 72 °Cfor 15 s; 
5) Measure the fluorescence of each sample. 
*Adjust temp for the specific primer pair.

SECTION: Part 1 - PCR Amplification - Step 2: Amplify the template
4. Repeat cycle steps 2 through 5 from step 3 until the rate at which the sample amplification decreases and starts to reach a plateau.
SECTION: Part 1 - PCR Amplification - Step 2: Amplify the template
5. Remove samples during the 72 °C.  Since the oligo pool consists of a complex number of sequences, samples removed outside of the elongation step will likely lead to hybridization with complement mismatches.  The simplest method would be to run 10-14 rounds of PCR amplification and allow the instrument to complete the final round.  If it is uncertain how many rounds you need to run for a particular oligo pool, you can set the instrument for an estimated number of rounds and then stop the round at any 72°C elongation step.  

SECTION: Part 1 - PCR Amplification - Step 3: Purify the template.
6. Column purify to remove enzyme, nucleotides, and primers.
In a 15 mL Falcon tube, mix the following:
800 µL  generated in Step 5;
4 mL .

SECTION: Part 1 - PCR Amplification - Step 3: Purify the template.
7. Run this mixture across a 100-µg capacity column using either a vacuum manifold.
SECTION: Part 1 - PCR Amplification - Step 3: Purify the template.
8. Wash the column twice with 300 µL , spinning the column in a table top centrifuge at maximum speed for 30 s each time.

SECTION: Part 1 - PCR Amplification - Step 3: Purify the template.
9. Elute the template by adding 170 µL  to the column, transferring the column to a fresh 1.7 mL Eppendorf tube, and spinning at maximum speed for 30 s.
SECTION: Part 1 - PCR Amplification - Step 3: Purify the template.
10. Set aside 10 µL  for quality control.

SECTION: Part 1 - PCR Amplification - Step 4: (Optional) Quality control for template reaction.
11. Two quality control steps can be performed to verify the quality of your PCR product.
1. Using a spectrophotometer, such as the Nanodrop or similar, measure the concentration of dsDNA in your product. The concentration should be between 0.01 µg/µL to 0.05 µg/µL.
2. The second quality control step is gel electrophoresis and will be described in part 4, step 5 (>>step 36 below).
SECTION: Part 2 – In-Vitro Transcription
12. The second step of this protocol is a high yield in-vitro transcription reaction which amplifies each PCR product multiple times while converting the product to RNA complements.  
SECTION: Part 2 – In-Vitro Transcription -  Step 1: In-vitro transcription.
13. In a fresh 1.7 mL Eppendorf tube, mix the following:
160 µL  created in Part 1.
176 µL 
250 µL  provided with the Quick HiScribe T7 polymerase kit
25 µL 
25 µL  (from the same HiScribe kit)

SECTION: Part 2 – In-Vitro Transcription -  Step 1: In-vitro transcription.
14. Incubate the reaction in a 37 °C incubator or dry bath for 720 min–960 min hours. While the reaction is complete after 360 min -480 min  hours, it is convenient to leave this reaction  . Remove 20 µL for quality control.

SECTION: Part 2 – In-Vitro Transcription - Step 2: (Optional) Quality control for the in vitro transcription.
15. To confirm that the in vitro transcription was successful, column purify the reaction then measure its concentration with a spectrophotometer.  

To purify, mix the following:
- 20 µL 
- 30 µL 
- 100 µL
- 150 µL . 

SECTION: Part 2 – In-Vitro Transcription - Step 2: (Optional) Quality control for the in vitro transcription.
16. Pass across a 100-ug-capacity spin column in a table-top centrifuge.
SECTION: Part 2 – In-Vitro Transcription - Step 2: (Optional) Quality control for the in vitro transcription.
17. Wash this column once with 400 µL , centrifuge 30 s top speed.
SECTION: Part 2 – In-Vitro Transcription - Step 2: (Optional) Quality control for the in vitro transcription.
18. Wash twice with 200 µL , centrifuge 30 s top speed.
SECTION: Part 2 – In-Vitro Transcription - Step 2: (Optional) Quality control for the in vitro transcription.
19. Elute the RNA with 100 µL .

SECTION: Part 2 – In-Vitro Transcription - Step 2: (Optional) Quality control for the in vitro transcription.
20. If successful, the concentration of the in vitro transcription should be between 0.5 µg/µL to 2 µg/µL.
SECTION: Part 2 – In-Vitro Transcription - Step 2: (Optional) Quality control for the in vitro transcription.
21. Purified RNA can also be run on a gel as described in Part 4, Step 5 (>>step 36 below).
SECTION: Part 3 - Reverse Transcription RNA to DNA
22. In this step of the protocol, our RNA molecules are used as Reverse Transcription templates to convert the large quantities of RNA produced by the high yield in vitro transcription to single-stranded DNA. To cleave off the 5' priming region and shorten the final probe, we use a uracil-modified reverse transcription primer for the reverse transcription reaction followed by use of a Uracil-Specfic Excision Reagent (USER Enzyme) to cleave the uracil nucleotide and remove the primer. The RNA template is then removed via alkaline hydrolysis, and the final encoding probes are purified and concentrated.  
SECTION: Part 3 - Reverse Transcription RNA to DNA - Step 1: Reverse transcription
23. To the unpurified in vitro transcription created in Part 2, add the following and mix well:
- 200 µL
- 120 µL  with uracil modification.
- 240 µL
- 24 µL
- 24 µL .

SECTION: Part 3 - Reverse Transcription RNA to DNA - Step 1: Reverse transcription
24. Incubate in a 50 °C  for 60 min hour. It is important to use a water bath, not an air incubator, to ensure that the temperature of the sample rises to 50 °C quickly.

SECTION: Part 4 – USER Enzyme Digest &amp; Purification of Encoding Probes - Step 1: USER Enzyme Digest
25. To digest, use a 1:20 (vol/vol)  and incubate at 37 °C for 1440 min to cleave off the priming region at the site of the uracil. 
SECTION: Part 4 – USER Enzyme Digest &amp; Purification of Encoding Probes - Step 2: Alkaline Hydrolysis
26. Split the above reaction into two 1.7-mL Eppendorf tubes and add the following to each:
-  300 µL
- 300 µL 

SECTION: Part 4 – USER Enzyme Digest &amp; Purification of Encoding Probes - Step 2: Alkaline Hydrolysis
27. Incubate in a 95 °C  for 15 min.

SECTION: Part 4 – USER Enzyme Digest &amp; Purification of Encoding Probes -  Step 3: Purification of ssDNA Product
28. Combine the two aliquots above into a single 50 mL Falcon tube and add the following:
- 4.8 mL 
- 19.2 mL .

SECTION: Part 4 – USER Enzyme Digest &amp; Purification of Encoding Probes -  Step 3: Purification of ssDNA Product
29. Mix well and split equally between eight 100-µg capacity spin columns.
SECTION: Part 4 – USER Enzyme Digest &amp; Purification of Encoding Probes -  Step 3: Purification of ssDNA Product
30. Pull the sample across the columns with a vacuum manifold or via centrifugation.
SECTION: Part 4 – USER Enzyme Digest &amp; Purification of Encoding Probes -  Step 3: Purification of ssDNA Product
31. Wash the columns once with750 µL.  Centrifuge to remove all of the wash buffer.  You may need to centrifuge this twice to fully remove all of the wash buffer.  

SECTION: Part 4 – USER Enzyme Digest &amp; Purification of Encoding Probes -  Step 3: Purification of ssDNA Product
32. Elute the columns using 100 µL .

SECTION: Part 4 – USER Enzyme Digest &amp; Purification of Encoding Probes -  Step 3: Purification of ssDNA Product
33. Combine eluates and set aside 10 µL for quality control.
SECTION: Part 4 – USER Enzyme Digest &amp; Purification of Encoding Probes -  Step 4: Concentration of Probe
34. Use a vacuum concentrator to dry the samples.  This process could take several hours.  Re-suspend the dried pellet in 24 µL  or if desired, hybridization buffer.  Store probe at -20 °C and avoid unnecessary Freeze-thaw cycles.

SECTION: Part 4 – USER Enzyme Digest &amp; Purification of Encoding Probes -  Step 4: Concentration of Probe
35. If vacuum concentrator isn’t available, concentrate probe using ethanol precipitation (refer to SOP009).
SECTION: Part 4 – USER Enzyme Digest &amp; Purification of Encoding Probes -  Step 4: Concentration of Probe
36. Quality Control.  Analyze the concentration and the purity of your finalized probe sample on a Nanodrop spectophotometer or similar.  Your ssDNA concentration should be around 34µg/µL.  
SECTION: Part 4 – USER Enzyme Digest &amp; Purification of Encoding Probes - Step 5: (Optional) Quality control of in vitro template, RNA, and probe.
37. We recommend running your PCR product, IVT product and your final probe product on a 15% TBE-urea polyacrylamide gel to review the size and quality of your products. Smearing can indicate degradation of your product, particularly for RNA molecules.  You should also note the size of the bands your are expecting to see and compare each band to one another.  IVT will shorten your product by 20nt removing the 3' primer as the reaction only occurs after the T7 promoter region at the 3' end of the probe.  Additionally, the USER enzyme digested product should be approximately 20nt shorter as this removes the 5' priming region.  These changes in the product lengths should be visible when running your gel.