TITLE: Enzymatic padlock probe preparation
AUTHORS: Chien-Ju Chen, Kian Kalhor, Kun Zhang

[DESCRIPTION]
This protocol accompanies the manuscript Mapping Human Tissues with Highly Multiplexed RNA in situ Hybridization (https://doi.org/10.1101/2023.08.16.553610). It protocol outlines the steps for the enzymatic amplification and preparation of padlock probes from an oligo pool. This strategy has a lower upfront cost compared to individual synthesis of probes and thus permits the use of tens of thousands of padlock probes per probe set. Additionally, the oligo pool serves as an unlimited source of padlock probes, as it can be expanded by PCR amplification upon need. 

[BEFORE_START]
The main starting material is an oligo pool with probe-set specific amplification primers on the 5' and 3' ends. In the example below, AP1V7 and AP2V7 are the primer pair that specifically amplify our probe set of interest.

[STEPS]
SECTION: PCR mass production
3. Prepare PCR production mix in a 15mL tube and add load 100 µL  to each well of a 96-well plate

 
run PCR program to amplify the oligo pool 
98 °C , 1 min -> (98 °C , 30 s ->55 °C 45 s ->72 °C 45 s )x14 cycles ->72 °C , 2 min -> 4 °C , hold

SECTION: oligo pool pre-amplification
1. Resuspend the oligo pool in water to a concentration of 7.02 ng/uL. Dilute the pool 100 times and run PCR to amplify the brain probe set.

 

PCR program: 
95 °C , 30 s -> (95 °C , 30 s ->55 °C 45 s ->72 °C 45 s )x17 cycles ->72 °C , 2 min -> 15 °C , hold
SECTION: PCR mass production
4. Pool the PCR production mix from the 96-well plate and prepare the ethanol precipitation mix in 5mL-tubes. (One 96-well plate can split into 8 5mL-tubes)

 
Mix the tubes by shaking and incubate them in a -80 °C freezer for 60 min.
Centrifuge the tubes at 4,000rpm, 4 °C  for 30 min  and discard the supernatant
SECTION: PCR mass production
5. Add 800 µL to each 5mL tube and transfer the solution into another 1.5mL tube. (8 5mL-tubes transfer into 8 1.5mL tubes).
Centrifuge at 14,000rpm, 4 °C  for 5 min  and discard the supernatant.
SECTION: PCR mass production
6. Air-dry the DNA pallets on ice bucket in AirClean hood for 10 min 
Rehydrate DNA pallets with 50 µL 
Pool the DNA solutions and use 8 QIAquick columns to purify.
Use Nanodrop to quantify the concentration.
Aliquot 3uL for TBU gel check
6. 
SECTION: Lambda Exo digestion
7. Prepare Lambda Exo digestion mix and split it into PCR tubes (100 µL per tube). Adjust the volume of DNA amplicon and ultrapure water so that the DNA amplicon per tube is around 5 µg 

 
Run PCR program: 37 °C ,120 min ->4 °C ,hold

SECTION: Lambda Exo digestion
8. Use 16 ssDNA/RNA Zymo-spin columns to purify the Lambda Exo-digested products and elute the columns with 50 µL 
Use Nanodrop to quantify the concentration.
Aliquot 3uL for TBU gel check
SECTION: USER digestion
9. Prepare USER digestion mix and split into PCR tubes (80 µL per tube). Adjust the volume of ssDNA and ultrapure water so that the ssDNA per tube is less than 2 µg .

 
Run PCR program: 37 °C  ,180 min ->4 °C ,hold.
Aliquot 3uL for TBU gel check
SECTION: DpnII digestion
10. Prepare DpnII oligo mix and add 15 µL  to each PCR tube
 
Run PCR program: 94 °C ,2 min -> 37 °C ,3 min -> 4 °C , hold

SECTION: DpnII digestion
11. Add 5 µL to each PCR tube (total volume is 100 µL per tube) and mix by pipetting.

Run PCR program:37 °C ,300 min ->4 °C , hold

SECTION: DpnII digestion
12. Use ssDNA/RNA Zymo-spin columns to purify each PCR tube and elute each column with 50 µL 
Use Nanodrop to quantify the concentration
Aliquot 3uL for TBU gel check

SECTION: TBU gel check (optional)
13. Pre-run one 12-well TBU-gel for at least 10 min 

Prepare TBU gel check samples
 
Notes: If PCR product and Lambda Exo sample look overloaded, dilute the sample by 10-20 times before TBU gel check.

Incubate TBU gel check samples at 70 °C for5 min  and immediately put on ice for 5 min 

Flush each well with TBE running buffer to remove excessive urea in the 12-well gel

Load the TBU gel check samples to the 12-well TBU gel

Run gel electrophoresis at 220-240 V for 20 min 
Example TBU gel for a padlock probe set with the final size of 150nt:
  

SECTION: Size selection
14. Pre-run 1-well TBU gels for at least 10 min 

Prepare enzyme-digested probes: add 180 µL to  180 µL 

Incubate the samples at 70 °C for 5 min and immediately put on ice for 5 min 

Flush each well with TBE buffer to remove excessive urea in each well.
SECTION: Size selection
15. Load samples to 1-well TBU gel.

Notes: Cap the amount of enzyme-digested probes per 1-well TBU gel at 2 µg  to avoid overloading and add the equivalent volume of 2x Novex buffer.  The rest of the enzyme-digested probes can be stored at -20 °C  freezer for the next round of size selection.

Run gel electrophoresis at 220-240 V for 20 min 

SECTION: Size selection
16. Cut enzyme-digested probes at the right size and transfer the cut-out gels to 0.5mL tubes (with holes at the bottom) on top of 2mL tubes

Centrifuge the tubes at 15,000rpm for 5 min to shred gels

Transfer the remaining gel in the 0.5mL tubes to the 2 mL tubes 
SECTION: Size selection
17. Add 450 µL to each 2mL tubes and mix the tubes by shaking

Freeze and thaw the solution in the -80 °C freezer,    and vortex the tubes in a 37 °C  incubator for 120 min 

SECTION: Size selection
18. Centrifuge the tubes at 15,000rpm, Room temperature  for 3 min 

Transfer the clear supernatant to Nanosep columns and centrifuge at 15,000 rpm, Room temperature for 3 min 

Repeat Nanosep filtering once and transfer the flow-through to 1.5 mL tubes

SECTION: Ethanol precipitation
19. Add 1 mL , 1.4 µL , 45 µL to each tube and mix by shaking
SECTION: Ethanol precipitation
20. Precipitate probes in a -80 °C freezer for at least 60 min 

Centrifuge at 10,000 rpm, 4 °C for 30 min  and discard the supernatant

SECTION: Ethanol precipitation
21. Wash the pallets with 750 µL  and centrifuge at 14,000rpm at 4 °C for 5 min 

SECTION: Ethanol precipitation
22. Discard the supernatant and air-dry the pallets in AirClean hood for 10 min 

SECTION: oligo pool pre-amplification
2. Purify the PCR product by QIAquick PCR purification kit and elute each QIAquick column with 50 µL 

Use Qubit with HS dsDNA kit to quantify the concentration and dilute the PCR product to 10nM by adding ultrapure water. This is called "10nM oligo pool 1st amplicon" in the following section.
SECTION: Ethanol precipitation
23. Rehydrate the pallets with 10 µL per tube and pool the probe solution together

Use Qubit with ssDNA kit to quantify the concentration of probe solution