TITLE: MPRA plasmid pool preparation
AUTHORS: Gerald Raffl, Boyan  Bonev

[DESCRIPTION]
Protocol to generate the MPRA plasmid pool.

[STEPS]
SECTION: PCR amplification of oligos
1. Design of single stranded oligo library:
5'-AGGACCGGATCAACT**CRE_270bp**CATTGCGTGAACCGA-3'
SECTION: PCR amplification of oligos
3. To transform the single stranded oligos into double stranded DNA, set up the following PCR reaction:
2 µL single stranded oligos 
5 µL primer pool_amp_F (10µM stock)
5 µL primer pool_amp_R (10µM stock)
50 µL NEBNext Ultra II Q5 Master Mix
38 µL nuclease free water
Split the reaction mix into 4 PCR tubes to avoid PCR jackpotting.

SECTION: PCR amplification of oligos
4. Incubate in a PCR cycler with heated lid:
30s - 98°C
8 cycles (10s - 98°C; 20s - 60°C; 30s - 72°C)
5min - 72°C
hold - 4°C
SECTION: PCR amplification of oligos
5. Combine the reaction mix from the 4 PCR tubes in a DNA low binding 1.5 tube. Clean up the PCR product with 1.8X vol. of AMPure XP magnetic beads following the manufacturer's protocol.
Elute the PCR product in 21 µL nuclease free water.
SECTION: PCR amplification of oligos
6. Determine the DNA concentration by measuring 1 µL on the Nanodrop.
SECTION: PCR amplification of oligos
7. Gibson overhangs and a 12bp barcode are added by PCR.
Set up the following PCR reaction:
variable: 25 ng PCR product from step   
5 µL primer CRS_pMPRA1-GibOH_F (10µM stock)
5 µL primer CRS_GibOH_uniRV_BC12_R (10µM stock)
50 µL NEBNext Ultra II Q5 Master Mix
to 100 µL: nuclease free water
Split the reaction mix into 4 PCR tubes to avoid PCR jackpotting.

SECTION: PCR amplification of oligos
8. Incubate in a PCR cycler with heated lid:
30s - 98°C
14 cycles (10s - 98°C; 20s - 60°C; 30s - 72°C)
5min - 72°C
hold - 4°C
SECTION: PCR amplification of oligos
9. Combine the reaction mix from the 4 PCR tubes and add 20 µL 6X gel loading dye. Perform DNA gel electrophoresis (30 min, 100 V) on a gel made of 1 % agarose in 1X TAE, with 1:30'000 SYBR Safe stain. Cut the band(s) at 399 bp using a blue light illuminator.
SECTION: PCR amplification of oligos
10. Extract DNA from the agarose by using the Zymoclean Gel DNA Recovery Kit and following the manufacturer's protocol. Elute in 20 µL nuclease free water. 
SECTION: PCR amplification of oligos
11. Repurify the product using the DNA Clean & Concentrator Kit (binding buffer ratio 5:1) and following the manufacturer's protocol. Elute in 12 µL nuclease free water. 
SECTION: PCR amplification of oligos
12. Determine the DNA concentration by measuring 1 µL on the Nanodrop.
This is the insert library for the subsequent Gibson assembly. Freeze at -20°C until use.
SECTION: Amplification of the backbone
13. To obtain more backbone, multiple reactions can be set up in parallel.
Set up the following PCR reaction:
1 ng pMPRA1 (addgene #49349)
2.5 µL pMPRA1_BB_F (10µM stock)
2.5 µL pMPRA1_BB_R (10µM stock)
25 µL Q5 Hot Start High-Fidelity 2X Master Mix
to 50 µL: nuclease free water
SECTION: Amplification of the backbone
14. Incubate in a PCR cycler with heated  lid:
30s - 98°C
35 cycles (10s - 98°C; 20s - 68°C; 1min - 72°C)
5min - 72°C
hold - 4°C
SECTION: Amplification of the backbone
16. Determine DNA concentration by measuring 1 µL on the Nanodrop.
This is the backbone for the subsequent Gibson assembly. Freeze at -20°C until use.
SECTION: Gibson assembly
18. Set up a Gibson assembly reaction in a PCR tube:
1 µg backbone from step   
2:1 molar ratio (~ 320 ng) insert from step   
to 50 µL: nuclease free water

SECTION: Gibson assembly
19. Add 50 µL NEBuilder HiFi DNA Assembly Master Mix, mix briefly by pipetting up and down and immediately transfer reaction to the PCR cycler at 50°C.
Incubate for 1h at 50°C
SECTION: Gibson assembly
17. Preheat a PCR cycler to 50°C, with lid temperature ≥ 75°C
SECTION: Gibson assembly
20. Purify the product using the DNA Clean & Concentrator Kit (binding buffer ratio 2:1) and following the manufacturer's protocol. Elute in 9 µL nuclease free water. 

SECTION: Amplification of the backbone
15. Perform DNA gel electrophoresis and DNA gel extraction (band at 2499bp) as described in steps   -   . Elute in 20 µL nuclease free water.

SECTION: Gibson assembly
21. Determine DNA concentration by measuring 1 µL on the Nanodrop.
Ideally, continue with electrotransformation straight away.
SECTION: Electrotransformation &amp; MPRA pre-pool preparation
22. Prewarm 1 mL SOC medium, ideally to 37°C.
Keep a 1.5 mL Eppendorf tube warm.
Thaw a vial (100 µL) of ElectroMAX Stbl4 Competent Cells on ice.
Precool four electroporation cuvettes on ice.

SECTION: Electrotransformation &amp; MPRA pre-pool preparation
23. Set electroporator to the following parameters:
1.8 kV
25 μF
200 Ω
SECTION: Electrotransformation &amp; MPRA pre-pool preparation
24. Mix bacteria with 400 ng of purified Gibson assembly product from step   .
Distribute bacteria to the 4 cuvettes (25 µL each), while keeping them on ice.

SECTION: Electrotransformation &amp; MPRA pre-pool preparation
25. Electroporate each cuvette with parameters set in step    and immediately add 200 µL warm SOC medium.

SECTION: Electrotransformation &amp; MPRA pre-pool preparation
26. Combine the 4 reactions in the 1.5 mL Eppendorf tube. 
Shake the electrotransformed bacteria for 30min at 37°C and 200 rpm.
SECTION: Electrotransformation &amp; MPRA pre-pool preparation
27. In the meantime, prewarm 24 LB-agar plates (10 cm diameter) with 100 µg/mL Carbenicillin to 37°C.
SECTION: Electrotransformation &amp; MPRA pre-pool preparation
28. Vortex bacteria thoroughly and prepare the following 2 conditions:
condition A: 700 µL bacteria + 800 µL warm LB medium
condition B:  70 µL bacteria + 1430 µL warm LB medium

SECTION: Electrotransformation &amp; MPRA pre-pool preparation
29. From both conditions, prepare dilutions for counting plates:
1:1K dilution (7 µL bacteria + 693 µL warm LB medium)
1:10K dilution (70 µL bacteria [1:1K dilution] + 630 µL warm LB medium)
When plating 140 µL, the counting plate has 1'000 times / 10'000 times less colonies than all 10 plates of a certain condition combined.
SECTION: Electrotransformation &amp; MPRA pre-pool preparation
30. For each condition:
Plate 140 µL for both counting plates
Plate 140 µL bacteria mix from step   on 10 plates.

SECTION: Electrotransformation &amp; MPRA pre-pool preparation
31. Label all plates and incubate them at 37°C overnight (min. 16h).
SECTION: Electrotransformation &amp; MPRA pre-pool preparation
33. Depending on the number of CREs in the pool, and the desired number of barcodes per CRE; calculate the required colony number using the following formula: 

required colony number = (number of CREs) * (desired number of barcodes per CRE)

Based on the required colony number, choose a condition (undiluted or 1:10 diluted) and scrape the respective number of colonies using cell scrapers and LB medium.
SECTION: Electrotransformation &amp; MPRA pre-pool preparation
32. Count colonies on counting plates and calculate the number of transformants for each condition. If there is too many colonies, count a quarter of the plate and multiply the count by 4.
 

Don't trash the counting plates yet.
SECTION: Electrotransformation &amp; MPRA pre-pool preparation
34. Combine the scraped colonies in one tube and vortex to resuspend. 
Extract plasmid DNA using the QIAprep Spin Miniprep Kit and following the manufacturer's protocol. Depending on the amount of bacteria, split the mixture into multiple reactions.
SECTION: Electrotransformation &amp; MPRA pre-pool preparation
35. Determine DNA concentration by measuring 1 µL on the Nanodrop.
Plasmid DNA can be stored at -20°C for months.
This is the MPRA pre-pool which will be used for:
CRE-Barcode-association sequencing
Subsequent cloning: insertion of minimal promoter and reporter gene


SECTION: Electrotransformation &amp; MPRA pre-pool preparation
36. To verify the correct identity of the MPRA pre-pool, perform analytical restriction digest with 250 ng of the MPRA pre-pool. 
Set up two separate reactions, following the manufacturer's protocol:
Linearization with KpnI
Cut out CRE insert with KpnI and SpeI
SECTION: Electrotransformation &amp; MPRA pre-pool preparation
37. Perform DNA gel electrophoresis as described in step   . 
 

SECTION: Restriction digest &amp; ligation of MP_mScarlet-I
40. In this section, the minimal promoter and reporter gene (mScarlet-I) are added to the MPRA pre-pool via restriction cloning.

The plasmid pGR029 (note: also pGR023 and pGR025 - pGR033) contains 
the minimal Promoter (derived from pNL3.1[Nluc/minP], Promega) and 
mScarlet-I (kind gift from the Goetz Lab)
flanked by KpnI and EcoRI restriction sites.

Both pGR029 and the MPRA pre-pool are digested with EcoRI and KpnI. For each reaction, use 5 µg input DNA and 2.5 µL of both enzymes in 50 µL reactions, shaking for 2h at 37°C.
SECTION: Restriction digest &amp; ligation of MP_mScarlet-I
41. Perform DNA gel electrophoresis on the pGR029 digest as described in step   , and cut the relevant band at 779bp.
 


SECTION: Restriction digest &amp; ligation of MP_mScarlet-I
42. Extract DNA from the agarose by using the Zymoclean Gel DNA Recovery Kit and following the manufacturer's protocol. Elute in 20 µL nuclease free water. 
SECTION: Restriction digest &amp; ligation of MP_mScarlet-I
43. Repurify the gel-extracted product using the DNA Clean & Concentrator Kit (binding buffer ratio 5:1) and following the manufacturer's protocol. Elute in 12 µL nuclease free water. This is the insert for the subsequent ligation reaction.
SECTION: Restriction digest &amp; ligation of MP_mScarlet-I
44. Purify the restriction digest of the MPRA pre-pool using the DNA Clean & Concentrator Kit, and elute in 12 µL nuclease free water. This is the backbone for the subsequent ligation reaction.
SECTION: Restriction digest &amp; ligation of MP_mScarlet-I
45. Determine the DNA concentrations by measuring 1 µL on the Nanodrop.
Freeze at -20°C until use.
SECTION: Restriction digest &amp; ligation of MP_mScarlet-I
46. Set up the following ligation reaction:
2 µg backbone (digested MPRA pre-pool)
2:1 molar ratio of insert (MP_mScarlet-I) => ~ 550 ng
to 175 µL: nuclease free water
20 µL T4 DNA ligase buffer (10X)
5 µL T4 DNA ligase
SECTION: Electrotransformation &amp; MPRA pre-pool preparation
38. To verify the correct identity of individual clones, pick 16 colonies from a counting plate and inoculate overnight cultures (2 mL LB medium with 100 µg/mL Carbenicillin, shaking at 37°C overnight).
SECTION: Electrotransformation &amp; MPRA pre-pool preparation
39. Extract plasmid DNA from individual clones using the QIAprep Spin Miniprep Kit and send plasmids for Sanger sequencing using the following primers: RVprimer3(pMPRA1)_F and EBV-rev(pMPRA1)_R
 

SECTION: Restriction digest &amp; ligation of MP_mScarlet-I
47. Incubate in this order: 30min at 4°C, 4h at 16°C and 30min at RT
SECTION: Restriction digest &amp; ligation of MP_mScarlet-I
48. Purify the ligation product using the DNA Clean & Concentrator Kit (binding buffer ratio 2:1) and following the manufacturer's protocol. Elute in 9 µL nuclease free water. 
SECTION: Restriction digest &amp; ligation of MP_mScarlet-I
49. Determine DNA concentration by measuring 1 µL on the Nanodrop.
SECTION: Electrotransformation &amp; MPRA pool preparation
50. Perform electrotransformation of 100 µL electrocompetent bacteria with 400 ng of purified ligation product as described in   -   .

SECTION: Electrotransformation &amp; MPRA pool preparation
51. In the meantime, prewarm 3 normal LB-agar plates (10 cm diameter) and 10 big LB-agar plates (15 cm diameter) with 100 µg/mL Carbenicillin to 37°C.
SECTION: Electrotransformation &amp; MPRA pool preparation
52. Fill the bacterial culture up to 4 mL with LB medium. 
SECTION: Electrotransformation &amp; MPRA pool preparation
53. Prepare dilutions for counting plates:
1:1K:   5 µL bacterial culture + 195 µL LB medium
1:10K:   20 µL (1:1K dil.) + 180 µL LB medium
1:100K:   20 µL (1:10K dil.) + 180 µL LB medium

SECTION: Electrotransformation &amp; MPRA pool preparation
54. Plate 140 µL from each counting plate dilution on LB-agar plates (10 cm diameter).
Plate 350 µL of the bacterial culture from step    on each of the 10 big LB-agar plates (15 cm diameter).

SECTION: Electrotransformation &amp; MPRA pool preparation
55. Label all plates and incubate them at 37°C overnight (20h).
SECTION: Electrotransformation &amp; MPRA pool preparation
56. Count colonies on counting plates and calculate the number of transformants. If there is too many colonies, count a quarter of the plate and multiply the count by 4.

To preserve library complexity, the number of transformants should exceed the desired library complexity from step   .

SECTION: Electrotransformation &amp; MPRA pool preparation
57. Scrape all colonies from the 10 big LB-agar plates using cell scrapers and LB medium. Combine the bacteria and vortex thoroughly to resuspend.
SECTION: Electrotransformation &amp; MPRA pool preparation
58. Extract plasmid DNA using the EndoFree Plasmid Maxi Kit and following the manufacturer's protocol. In the final step, resuspend the DNA pellet in 1X sterile PBS.
SECTION: Electrotransformation &amp; MPRA pool preparation
59. Determine DNA concentration by measuring 1 µL on the Nanodrop.
This is the final MPRA pool. Freeze in aliquots (!) at -20°C. Repeated freeze-thaw cycles are not recommended!
SECTION: Electrotransformation &amp; MPRA pool preparation
60. To verify the correct identity of the MPRA pool, perform analytical restriction digest with 250 ng of the MPRA pool. 
Set up two separate reactions, following the manufacturer's protocol:
Linearization with BamHI
BamHI and KpnI
SECTION: Electrotransformation &amp; MPRA pool preparation
61. Perform DNA gel electrophoresis as described in step   . 
 


SECTION: PCR amplification of oligos
2. Resuspend the single stranded oligos in nuclease free water to obtain a concentration of 20 ng/µL.