TITLE: MPRA library preparation
AUTHORS: Gerald Raffl, Boyan  Bonev

[DESCRIPTION]
Protocol to generate both DNA and RNA MPRA libraries. 

[STEPS]
SECTION: DNA &amp; RNA extraction from fixed nuclei
2. Pellet sorted nuclei in a 1.5 mL tube at 2500 x g, 4°C for 3min.
Resuspend pellet in 1X ProtK digestion buffer (95 µL nuclease free water, 95 µL 2X digestion buffer, 10 µL Proteinase K)
SECTION: DNA &amp; RNA extraction from fixed nuclei
1. Preheat a ThermoMixer to 55°C.
SECTION: DNA &amp; RNA extraction from fixed nuclei
3. Incubate at 55°C, 600 rpm for 1h. (Proteinase K digestion)
In the meantime, preheat another ThermoMixer to 65°C.
SECTION: DNA &amp; RNA extraction from fixed nuclei
4. Incubate at 65°C, 600 rpm for 15min. (Reverse Crosslinking)
SECTION: DNA &amp; RNA extraction from fixed nuclei
5. Centrifuge at maximum speed for 3min. (Get rid of cell debris)
In the meantime, set the 2 ThermoMixers to 37°C and 26°C, respectively.
SECTION: DNA &amp; RNA extraction from fixed nuclei
7. Continue with the Quick-DNA/RNA Microprep Plus Kit following the manufacturer's protocol. 
Perform in-column DNase digest for the RNA samples.
SECTION: TURBO DNase digest and Reverse Transcription
10. Add to each RNA sample:
1.5 µL TURBO DNase buffer (10X)
1 µL TURBO DNase
Incubate at 37°C, 600rpm for 20min
SECTION: DNA &amp; RNA extraction from fixed nuclei
8. For DNA samples: 
Elute in 21 µL nuclease free water. 
Measure DNA concentration with the Qubit™ dsDNA HS Assay Kit, using 1 µL DNA.
Freeze at -20°C for later use.
SECTION: DNA &amp; RNA extraction from fixed nuclei
9. For RNA samples: 
Elute in 15 µL nuclease free water.
Continue with TURBO DNase digestion and Reverse Transcription.
SECTION: DNA &amp; RNA extraction from fixed nuclei
6. Transfer supernatant (190 µL) to a new tube, add 190 µL RNA/DNA lysis buffer (from Quick-DNA/RNA Microprep Plus Kit) and mix thoroughly.
SECTION: TURBO DNase digest and Reverse Transcription
11. Add 2 µL inactivation reagent to each sample.
Incubate at 26°C, 1600rpm for 5min.
SECTION: TURBO DNase digest and Reverse Transcription
12. Transfer each sample to 0.5 mL tubes (critical, allows better aspiration of supernatant).
Centrifuge at 10'000 x g for 90s.
SECTION: TURBO DNase digest and Reverse Transcription
13. Aspirate the supernatant (15 µL). Do not take up any inactivation reagent!
Measure RNA concentration with the Qubit™ RNA HS Assay Kit, using 1 µL RNA.
SECTION: TURBO DNase digest and Reverse Transcription
14. Optional: Determine the RIN value by analyzing the RNA samples with the RNA 6000 Pico Kit, following the manufacturer's protocol.
SECTION: TURBO DNase digest and Reverse Transcription
15. Use 12 µL RNA for Reverse Transcription. For that, set up the following reaction in 0.2 mL PCR tube:
12 µL RNA
1.5 µL dNTP mix
1.5 µL Oligo(dT)18 Primer 

SECTION: TURBO DNase digest and Reverse Transcription
16. Incubate in a thermocycler with heated lid:
5min at 65°C
SECTION: TURBO DNase digest and Reverse Transcription
17. Place reaction on ice for 1min.
Add 15 µL 2X RT mix to each sample (6 µL 5X RT buffer, 7.5 µL nuclease free water, 1 µL Maxima H- RT, 0.5 µL RNasin plus).
SECTION: TURBO DNase digest and Reverse Transcription
18. Incubate in a thermocycler with heated lid:
30min at 50°C
5min at 85°C
SECTION: TURBO DNase digest and Reverse Transcription
19. Transfer each sample to a DNA low binding 1.5 mL tube.
Clean up the cDNA with 1.5X vol. (= 45 µL) of AMPure XP magnetic beads following the manufacturer's protocol.
Elute the cleaned cDNA in 21 µL nuclease free water.
Freeze cDNA at -20°C for later use.
SECTION: Attaching UMIs
20. In the two subsequent sections, DNA sequencing libraries are generated from both DNA and cDNA (RNA) samples. This is done in the exact same way for both sample types. 

In this first PCR, UMIs are attached by 3 PCR cycles. Primer binding sites are in the C-terminal part of mScarlet-I (FWD_mScar_Tn7_10UMI_3) and immediately downstream of the 12bp barcode (RV_univ_MPRA). The fragment created in this PCR has a length of 196bp.

Set up the following PCR reaction:
17 µL DNA/cDNA from step    and step   , respectively
3 µL nuclease free water
2.5 µL primer FWD_mScar_Tn7_10UMI_3 (10µM stock)
2.5 µL primer RV_univ_MPRA (10µM stock)
25 µL NEBNext Ultra II Q5 Master Mix

 

SECTION: Attaching UMIs
21. Incubate in a PCR cycler with heated lid:
30s - 98°C
3 cycles (10s - 98°C; 30s - 65°C; 1min - 72°C)
5min - 72°C
hold - 4°C
SECTION: Attaching UMIs
22. Transfer each sample to a DNA low binding 1.5 mL tube.
Clean up each sample with 1.2X vol. (= 60 µL) of AMPure XP magnetic beads following the manufacturer's protocol.
Elute in 20 µL nuclease free water.
SECTION: Library amplification
23. The library amplification step is split up in 2 separate PCRs. In the first PCR, the P7 illumina adapter is added. Then, in the remaining cycles, also the P5 illumina adapter is added. By splitting up the library amplification in two PCRs (with a total of 20-30 cycles, depending on input), the likelihood of primer dimer formation is drastically reduced in low-input samples.

Set up the following PCR reaction:
20 µL sample from step  
2.5 µL primer Ad2.X_index (2µM stock)
2.5 µL primer RV_univ_MPRA (2µM stock)
25 µL NEBNext Ultra II Q5 Master Mix

SECTION: Library amplification
24. Incubate in a PCR cycler with heated lid:
30s - 98°C
X cycles (10s - 98°C; 90s - 65°C)
5min - 65°C
hold - 4°C
X cycles: 10 for DNA samples, 12 for cDNA samples
SECTION: Library amplification
25. Transfer each sample to a DNA low binding 1.5 mL tube.
Clean up each sample with 1.2X vol. (= 60 µL) of AMPure XP magnetic beads following the manufacturer's protocol.
Elute in 10 µL nuclease free water.
SECTION: Library amplification
26. Use 1.5 µL sample from step   for qPCR to estimate the remaining PCR cycles.
Set up the following qPCR reaction:
1.5 µL sample
0.5 µL primer Ad2.X_index (2µM stock)
0.5 µL primer P5NEXTPT5 (2µM stock)
2.5 µL nuclease free water
5 µL 2X Luna Universal qPCR Master Mix

SECTION: Library amplification
27. Incubate in a Real-Time thermocycler:
5mins - 95°C
45 cycles (10s - 98°C; 10s - 60°C; 20s - 72°C)
Cooling
SECTION: Library amplification
28. Determine the cycle number, where the amplification curve was at its half maximum. 
Subtract 1 from this number. This is the required remaining cycle number.
SECTION: Library amplification
29. Set up the following PCR reaction:
8 µL sample from step  
12 µL nuclease free water
2.5 µL primer Ad2.X_index (2µM stock)
2.5 µL primer P5NEXTPT5 (2µM stock)
25 µL NEBNext Ultra II Q5 Master Mix
SECTION: Library amplification
30. Incubate in a PCR cycler with heated lid:
30s - 98°C
X cycles (10s - 98°C; 90s - 65°C)
5min - 65°C
hold - 4°C
X cycles: number determined in step   

SECTION: Library amplification
31. Transfer each sample to a DNA low binding 1.5 mL tube.
Clean up each sample with 0.8X vol. (= 40 µL) of AMPure XP magnetic beads following the manufacturer's protocol.
 

Elute in 10 µL nuclease free water. This is the final library.
SECTION: Library amplification
32. Quantify by measuring 1 µL with the Qubit™ dsDNA HS Assay Kit.
Analyze the final library with the High Sensitivity DNA Kit on the Agilent 2100 Bioanalyzer.
 

SECTION: CRE-barcode association
33. In this section, the DNA sequencing library for the CRE-barcode association is created. For that, the MPRA pre-pool (without min.Promoter_mScarlet-I) is used as input.

Set up the following PCR reaction:
5 ng MPRA pre-pool
to 20 µL: nuclease free water
2.5 µL primer FWD_CRS_Tn7 (10µM stock)
2.5 µL primer RV_univ_MPRA (10µM stock)
25 µL NEBNext Ultra II Q5 Master Mix
SECTION: CRE-barcode association
34. Incubate in a PCR cycler with heated lid:
30s - 98°C
3 cycles (10s - 98°C; 30s - 65°C; 3min - 72°C)
5min - 72°C
hold - 4°C
 

SECTION: CRE-barcode association
35. Transfer the sample to a DNA low binding 1.5 mL tube.
Clean up the sample with 1.2X vol. (= 60 µL) of AMPure XP magnetic beads following the manufacturer's protocol.
Elute in 20 µL nuclease free water.
SECTION: CRE-barcode association
36. Set up the following PCR reaction:
20 µL PCR product
2.5 µL primer Ad2.X_index (2µM stock)
2.5 µL primer P5NEXTPT5 (2µM stock)
25 µL NEBNext Ultra II Q5 Master Mix
SECTION: CRE-barcode association
37. Transfer the sample to a DNA low binding 1.5 mL tube.
Clean up the sample by double size selection with 0.5X vol. and 0.8X vol. of AMPure XP magnetic beads following the manufacturer's protocol.
Elute in 10 µL nuclease free water.
SECTION: CRE-barcode association
38. Quantify by measuring 1 µL with the Qubit™ dsDNA HS Assay Kit.
Analyze the final library with the High Sensitivity DNA Kit on the Agilent 2100 Bioanalyzer.