TITLE: Saturation Mutagenesis-Reinforced Functional Assays (SMuRF)  for alpha-dystroglycan glycosylation enzymes (using FKRP and LARGE1 as examples)
AUTHORS: Kaiyue Ma, Shushu Huang, Jenny Xu, Angela Lek, Monkol Lek

[DESCRIPTION]


Interpretation of disease-causing genetic variants remains a challenge in the field of human
genetics and rare disease. Current costs and complexity of performing deep mutational
scanning for charting variant effects hampers crowd-sourcing approaches toward
genome-wide resolution of variants in all disease-related genes. Our framework,
Saturation Mutagenesis-Reinforced Functional assays (SMuRF), addresses these
issues by modularizing DMS components, offering simple and cost-effective
saturation mutagenesis, as well as streamlining functional assays to enhance
interpretation of unresolved variants. Applying SMuRF to neuromuscular disease
genes FKRP and LARGE1, we have generated functional scores for
over 99.8% of all possible coding single nucleotide variants (SNVs), providing
an additional line of evidence for clinical variant interpretation in
dystroglycanopathies. Data generated from SMuRF enables severity prediction, resolve
critical protein structural regions susceptible to missense disruptions, and
provide training datasets for development of computational predictors. In
summary, our approach provides a framework for enabling variant-to-function
insights for disease genes in a manner that is accessible for crowd-sourcing
implementation across standard research laboratories.




[STEPS]
SECTION: Create FKRP-KO and LARGE1-KO cell lines via CRISPR RNP nucleofection
1. Prepare RNP complexes in   

SECTION: Create FKRP-KO and LARGE1-KO cell lines via CRISPR RNP nucleofection
2. Transfer the mixed samples to the wells of the 16-well Nucleocuvette Strips
Perform nucleofection with   
Use EN-138 for HAP1; CA-137 for MB135
SECTION: Create FKRP-KO and LARGE1-KO cell lines via CRISPR RNP nucleofection
3. Allow the nucleofected cells to recover in the growth medium
Plate the nucleofected cells sparsely and allow them to form monoclonal clusters
Pick monoclonal cells under microscope

SECTION: Create FKRP-KO and LARGE1-KO cell lines via CRISPR RNP nucleofection
4. Determine indel event of each clonal line via targeted Sanger sequencing.
Primers used:
FKRP-GT-F: CATCACCCTCAACCTTCTGGTC
FKRP-GT-R: CATCAGGTACTAGGGCCACAAACTC
LARGE1-GT-F: GGCAATCGGGACTTTGGACA
LARGE1-GT-R: GCCTCGCCATGTAGTAAGGG



SECTION: Create FKRP-KO and LARGE1-KO cell lines via CRISPR RNP nucleofection
1.1. Combine 18 µL supplemented   , 6 µL of 30 micromolar (µM) sgRNA, and 1 µL of 20 micromolar (µM) Cas9 protein at Room temperature.  Incubate at Room temperature for 10 min.

SECTION: Create FKRP-KO and LARGE1-KO cell lines via CRISPR RNP nucleofection
1.2. Spin down 150k cells at 100 x g, 10 min, resuspend in 5 µL supplemented   .  Add the RNP complexes to the cells.

SECTION: Lentivirus packaging
5. Grow HEK293T cells to 90% confluency in a 10-cm dish with 10 mL HEK cell growth media.
SECTION: PALS-C cloning for saturation mutagenesis
14. Synthesize 64-bp ssDNA oligos for each variant of all possible CDS SNVs using Twist Bioscience
SECTION: Lentivirus packaging
6. Create the plasmid mixture and perform packaging

SECTION: Lentivirus packaging
7. Collect and concentrate packaged virus.

SECTION: Lentivirus packaging
7.1. Remove supernatant and filter through a  .

SECTION: Lentivirus packaging
8. Collect concentrated virus.
SECTION: Lentivirus packaging
9. Titrate lentivirus with   per manufacturer's instructions.  For long-term storage, store lentivirus in cryovials at -80 °C.

SECTION: Transduction
10. Plate cells to transduce in wells.
(Perform pre-experiments following the steps below to decide optimal MOI/drug concentration)

SECTION: Transduction
12. Add lentivirus for a spinfection at 800 x g, 60 min, 30 °C.

SECTION: Transduction
13. After one day, refresh medium and start drug selection if applicable. Perform drug selection for 7 days-14 days, using a well of un-transduced cells as a negative control.  

If construct contains BSD, add final concentration 5 µg/mL   .
If construct contains PuroR, add final concentration 1 µg/mL  .  

SECTION: PALS-C cloning for saturation mutagenesis
16. Anneal primers carrying degenerate nucleotides to the plasmid template and extend towards the 5' end. Conditions as below:

 

 

Notes:
Q5 polymerase is likely to be limiting factor--optimize volume if necessary.
Determine annealing temperature/elongation time based on the product that is most difficult to amplify.  If different blocks require vastly different conditions, multiple reactions can be performed.  70 °C and 40 s were used for FKRP, and 68 °C and 90 s were used for LARGE1.

SECTION: QC of plasmid pools and saturation mutagenesis
25. Perform QC on plasmid pools using GENEWIZ Amplicon-EZ service.
SECTION: Lentiviral packaging
26. Perform lentiviral packaging on one 10-cm dish of HEK293T cells.  Use small-scale pre-experiments to determine viral dosage for optimal separation
SECTION: Lentiviral packaging
27. Using GoStix Value as quantified by the  , scale the dosage for each block.

For each block, plate 600k HAP1 cells or 200k MB135 cells in a well of a 6-well plate.  After transduction and drug selection, for FACS, expand this number to 30M+.

Package lentiviral pools of all blocks at the same time using reagents and helper plasmids from the same batch to avoid batch effects.  Use 1e3 - 1e4 GV x µL of lentivirus per block. 
SECTION: Staining for FFC and FACS
28. Wash cells twice with   .

SECTION: Staining for FFC and FACS
29. Digest cells with   and count with an Automated Cell Counter (Bio-Rad, TC20).


SECTION: Staining for FFC and FACS
30. Spin down 30M cells at 700 x g, 15 min, 4 °C. Resuspend in 3 mL   supplemented with 30 µL .  

SECTION: Staining for FFC and FACS
32. Add 7 mL PEB buffer (1 volume of  , 19 volumes of  ) to the tube.

SECTION: Staining for FFC and FACS
33. Spin cells down at 700 x g, 15 min, 4 °C.

SECTION: Staining for FFC and FACS
34. Rock sample gently at room temperature for 30 min.

SECTION: Staining for FFC and FACS
35. Add 7 mL   . Spin cells down at 700 x g, 15 min, 4 °C. Resuspend in 3 mL MAGIC buffer (5% FBS; 0.1% NaAz w/v; 10% 10×  ;  ) supplemented with 1:200  (discontinued, or the same antibody made in Dr. Kevin Campbell's lab).

SECTION: Staining for FFC and FACS
36. Rock sample gently at 4 °C for 1200 min.

SECTION: Staining for FFC and FACS
37. Add 7 mL MAGIC buffer. 
.
SECTION: Staining for FFC and FACS
38. Rock sample gently at 4 °C for 1200 min in the dark.

SECTION: Staining for FFC and FACS
39. Add 7 mL   to the sample.  

SECTION: NGS library construction
40. Spin down the cells harvested from FACS at 800 x g, 10 min, 4 °C.

SECTION: NGS library construction
41. Harvest gDNA from each sample using  

SECTION: NGS library construction
42. Step 1: Use primers specific to the lentiviral backbone to amplify the lentiviral CDS sequences of each sample
SECTION: NGS library construction
43. Step 2: Isolate blocks.
SECTION: Immunofluorescence
45. Place   in a 24-well plate.

SECTION: Immunofluorescence
46. Coat coverslips in   and immediately remove. Air-dry.

SECTION: Immunofluorescence
47.1. Resuspend 250k MB135 cells in .5 mL growth medium (  with 20%  , 1x   , 51 ng/mL   , and 10 ng/mL  )

SECTION: Immunofluorescence
47.3. One day later, change out the medium for   with 1x   .  
SECTION: Immunofluorescence
48.1. Wash cells with  .

SECTION: Immunofluorescence
49. Block cells with 2 % (w/v)   in   at Room temperature for 60 min

SECTION: Immunofluorescence
50. Incubate with 1:200   in  2 % (w/v)   in    at 4 °C for 1200 min.

SECTION: Immunofluorescence
51. Wash cells in   and incubate in 1:100    in  2 % (w/v)   in    at Room temperature for 120 min. Keep cells in the dark.

SECTION: Immunofluorescence
52. Drop   onto  

SECTION: Immunofluorescence
53. Wash coverslips with  .   Place facedown on slides over drops of DAPI and keep at Room temperature in the dark for 30 min.

SECTION: Immunofluorescence
54. Image on a Revolve ECHO microscope
(DAPI - EX:380/30 EM:450/50 DM:425)
(FITC - EX:470/40 EM:525/50 DM:495)

SECTION: Packaging and infection of rVSV/ppVSV
55. Transfect HEK293T cells with LASV-GPC plasmid
SECTION: Packaging and infection of rVSV/ppVSV
56. Transduce cells with ppVSVΔG-VSV-G viral particles. Resulting particles will be referred to as ppVSV-LASV-GPC-Generation1.

SECTION: Packaging and infection of rVSV/ppVSV
57. Infect LASV-GPC transfected HEK293T cells with ppVSV-LASV-GPC-Generation1 to produce ppVSV-LASV-GPC-Generation2.  This reduces residual VSV-G in pseudotyped particles.  ppVSV-LASV-GPC-Generation2 used moving forward.

SECTION: Packaging and infection of rVSV/ppVSV
59. Determine MOI of ppVSV (performed at MOI 1-3)
SECTION: Packaging and infection of rVSV/ppVSV
60. Perform transduction and blasticidin drug selection as in FACS assay
SECTION: Packaging and infection of rVSV/ppVSV
61. Divide cells into 2 ~1M groups. Infect one group with rVSV at a concentration of 2e5 TCID50/mL (MOI ~0.5). Add 5 millimolar (mM) final concentration   during infection and recovery.

SECTION: Packaging and infection of rVSV/ppVSV
62. 3600 min later, refresh medium. Allow cells to recover for 720 min (cell count ~1M).

SECTION: Packaging and infection of rVSV/ppVSV
63. Harvest cells
SECTION: PALS-C cloning for saturation mutagenesis
17. Isolate products using block-specific primers.
SECTION: PALS-C cloning for saturation mutagenesis
18.     PALS-C Step 4: Add WT template and extend variant strands towards 3' end. 

 
     Q5 Rxn buffer      10 μL        Q5 Enh buffer      10 μL        10 mM dNTPs      1 μL        Purified Step3 product               Plasmid template               Q5 polymerase*      2 μL        Water      To 50 μL   

     98 °C       hot start        98 °C      5 min        72 °C      5 s        66 °C      20 s        72 °C      Elongation time*        12 °C      forever   

*Q5 polymerase is likely to be the limiting factor of Step4, volume of which requires optimization.
*Optional: the purpose is to enhance the annealing of all strands.
*The elongation time should be sufficient for the shortest strand to be elongated to the R2 primer site


SECTION: PALS-C cloning for saturation mutagenesis
20. Insert purified Step6 product into plasmid backbone.
SECTION: PALS-C cloning for saturation mutagenesis
21. Deliver Gibson assembly products to   via electrotransformation.

SECTION: PALS-C cloning for saturation mutagenesis
22. Seed all remaining bacteria in 150 mL LB broth with 100 µg/mL ampicillin.  Grow bacteria overnight. (Standard 37 °C 16hrs condition can be used but 30°C 20hrs is preferred)
SECTION: PALS-C cloning for saturation mutagenesis
23. Extract plasmid using  

SECTION: PALS-C cloning for saturation mutagenesis
24. Calculate colony forming units.
SECTION: PALS-C cloning for saturation mutagenesis
19. Perform electrophoresis and cut correct bands from gels.
IMPORTANT: Use 20 uL or less of water to dissolve after gel purification, or use vacuum concentrator to evaporate less water
SECTION: PALS-C cloning for saturation mutagenesis
20.1. PALS-C Step 7: Prepare backbone.

     XbaI      1.5 μL        BamHI-HF      1.5 μL        Plasmid template      3 μg        CutSmart      5 μL        Water      To 50 μL   

     37 °C (Lid: 60 °C)      40 min        12 °C      Forever   

SECTION: PALS-C cloning for saturation mutagenesis
20.2. Perform Gibson assembly.

     NEBuilderMaster Mix      20 μL        Backbone      210 ng        Purified Step6 product      140 ng        Water      To 30 μL   

     50 °C (Lid: 60 °C)      60 min        12 °C      Forever   

SECTION: PALS-C cloning for saturation mutagenesis
21.1. PALS-C Step 8: Assemble the following reaction for each block.

     Electrocompetent cells      40 μL        Assembly
  reaction      4 μL        Water      160 μL   

SECTION: PALS-C cloning for saturation mutagenesis
21.2. Split sample into 2 pre-chilled  

SECTION: PALS-C cloning for saturation mutagenesis
21.3. Use   @ 25 μF; 200 Ohms; 1800 volts. Avoid bubbles.



SECTION: PALS-C cloning for saturation mutagenesis
21.4. Add sample to  900 µL recovery media per cuvette. 
SECTION: PALS-C cloning for saturation mutagenesis
21.5. Combine transformed bacteria from both cuvettes in one tube. Shake at   .
SECTION: PALS-C cloning for saturation mutagenesis
21.6. Add 1/500 volume of the bacteria to 200 µL LB broth and plate it on an ampicillin LB agar plate for quick estimation of complexity


SECTION: QC of plasmid pools and saturation mutagenesis
25.1. For the plasmid pool of each FKRP block, perform the following reaction.
     Q5 Rxn buffer      10 μL        Q5 Enh buffer      10 μL        10 mM dNTPs      1 μL        Plasmid      ~300 ng        10 μM F primer      2.5 μL        10 μM R primer      2.5 μL        Q5 polymerase      0.5 μL        Water      To 50 μL   

     98 °C       hot start        98 °C      3 min        98 °C      6 s        70 °C      15 s        72 °C      5 s        Repeat 3-5 for 32 more cycles        72 °C      5 min        12 °C      Forever   

For the plasmid pool of each LARGE1 block, perform the following reaction:
     Q5 Rxn buffer      10 μL        Q5 Enh buffer      10 μL        10 mM dNTPs      1 μL        Plasmid      ~50 ng        10 μM F primer      2.5 μL        10 μM R primer      2.5 μL        Q5 polymerase      0.5 μL        Water      To 50 μL   

     98 °C       hot start        98 °C      3 min        98 °C      6 s        Annealing temperature      15 s        72 °C      7 s        Repeat 3-5 for 33 more cycles        72 °C      5 min        12 °C      Forever   

     Annealing temperature       Blocks        61 °C      1, 4, 6, 7        64 °C      5, 9, 10        66 °C      2, 3, 8   
Perform electrophoresis and gel purification (using   ). Send products for sequencing.

Mix purified products and perform the following reaction:
     Q5 Rxn buffer      10 μL        Q5 Enh buffer      10 μL        10 mM dNTPs      1 μL        Mixed purified products      100 ng        10 μM NGS-PCR3-F      2.5 μL        10 μM NGS-PCR3-R      2.5 μL        Q5 polymerase      1 μL        Water      To 50 μL   

     98 °C       hot start        98 °C      3 min        98 °C      6 s        72 °C      15 s        72 °C      8 s        Repeat 3-5 for 19 more cycles        72 °C      5 min        12 °C      Forever   
Perform PCR purification ( )and send sample for sequencing.

SECTION: NGS library construction
43.1. Perform the following reaction. See Supp. Method 8 for primers.
     Reagent      Catalog      Volume
  or weight        Q5
  Reaction Buffer      NEB,
  B9027SVIAL      10 µL        Q5 High
  GC Enhancer      NEB,
  B9028AVIAL      10 µL        10 mM
  dNTPs      NEB,
  N0447      1 µL        Q5 High-Fidelity
  DNA Polymerase      NEB,
  M0491SVIAL      1 µL        10µM
  PCR2-F             2.5 µL        10µM
  PCR2-R             2.5 µL        gDNA             0.2-0.5
  µg        Nuclease-Free
  Water             To 50 µL   

     Step1      98 °C      Hot
  start        Step2      98 °C      3 mins        Step3      98 °C      6 s        Step4      Annealing
  temperature      15 s        Step5      72 °C      7 s        Step
  3-5, 25 cycles        Step6      72 °C      5 mins        Step7      12 °C      Infinite   
Annealing temperatures:
     61 °C      LARGE1-blk1,
  LARGE1-blk4, LARGE1-blk6, LARGE1-blk7        64 °C      FKRP-blk1,
  LARGE1-blk5, LARGE1-blk9, LARGE1-blk10        66 °C      LARGE1-blk2,
  LARGE1-blk3, LARGE1-blk8        68 °C      FKRP-blk6        72 °C      FKRP-blk2,
  FKRP-blk3, FKRP-blk4, FKRP-blk5   

SECTION: NGS library construction
42.1. Perform the following reaction:
     PCR1-F      GATCGTCACTTGGTACCGGTTCTAGA        PCR1-R (FKRP)      TGGCACTTTTCGGGGGATCCTC        PCR1-R (LARGE1)      TGGCACTTTTCGGGGGATCCCT   

     Reagent      Catalog      Volume or weight        Q5 Reaction Buffer      NEB, B9027SVIAL      10 µL        Q5 High GC Enhancer      NEB, B9028AVIAL      10 µL        10 mM dNTPs      NEB, N0447      1 µL        Q5 High-Fidelity DNA Polymerase      NEB, M0491SVIAL      1 µL        10µM PCR1-F             2.5 µL        10µM PCR1-R             2.5 µL        gDNA             0.3-1 µg        Nuclease-Free Water             To 50 µL   

     Step1      98 °C      Hot start        Step2      98 °C      3 mins        Step3      98 °C      8 s        Step4      68 °C      20 s        Step5      72 °C      45 s        Step 3-5, 35 cycles        Step6      72 °C      5 mins        Step7      12 °C      Infinite   

SECTION: NGS library construction
42.2. Perform electrophoresis in 1% agarose gel. Expected band sizes are 1534 bp for FKRP and 2317 bp for LARGE1. Perform gel purification using   and elute in 25 µL nuclease-free water.

SECTION: NGS library construction
43.2. Perform PCR purification with  . Elute with 40 µL nuclease-free water. 

SECTION: NGS library construction
44. Perform adaptor addition.
SECTION: NGS library construction
44.1. Mix purified PCR2 products (200 ng each) and dilute to 11 ng/µL. Perform the following reaction:
     Reagent      Catalog      Volume
  or weight        Q5 Reaction
  Buffer      NEB,
  B9027SVIAL      10 µL        Q5 High
  GC Enhancer      NEB,
  B9028AVIAL      10 µL        10 mM
  dNTPs      NEB,
  N0447      1 µL        Q5
  High-Fidelity DNA Polymerase      NEB,
  M0491SVIAL      1 µL        10µM
  PCR3-F             2.5 µL        10µM
  PCR3-R             2.5 µL        Mixed sample             23 µL   

     Step1      98 °C      Hot
  start        Step2      98 °C      3 mins        Step3      98 °C      6 s        Step4      72 °C      15 s        Step5      72 °C      8 s        Step
  3-5, 25 cycles        Step6      72 °C      5 mins        Step7      12 °C      Infinite   
Set 3 * 50 µL reactions.

Perform   and elute with 50 µL nuclease free water.

SECTION: NGS library construction
44.2. Send PCR3 product for next generation sequencing and use GENEWIZ Amplicon-EZ service to check quality and coverage. Sequence using Hiseq X service.
SECTION: Staining for FFC and FACS
31. Perform all following steps in the dark. Rock sample for 30 min.

SECTION: PALS-C cloning for saturation mutagenesis
17.1. PALS-C step 2: use universal F1 primer and block-specific adaptor primer R1s to amplify variant strands using the conditions below. 

 

 
 
*The input should be decided based on the position of the block. The more distant a block is from the 5’ side, the more input is required. Evenly distributed input for all 6 blocks of FKRP generated enough yield for subsequent steps, while the 3’ side blocks of LARGE1 required extra input. Step1-2 should be repeated if product yield is insufficient for subsequent steps.

SECTION: PALS-C cloning for saturation mutagenesis
16.1. Perform PCR purification using   per manufacturer's instructions.

SECTION: PALS-C cloning for saturation mutagenesis
17.2. Perform PCR purification on 19.1 products using   .

SECTION: PALS-C cloning for saturation mutagenesis
17.3. PALS-C Step 3: Use type2S enzyme to remove the block-specific adaptor via restriction enzyme digest.
 
 
     Type2S enzyme*      2 μL        Purified Step2 product      1.2 μg        Reaction buffer      5 μL        Water      To 50 μL   

     Reaction temperature (Lid: 60 °C)      50 min        12 °C      Forever   
*BsmBI was used for FKRP and BsaI was used for
LARGE1. The type2S enzymes were picked to avoid the presence of their
recognition sites within the CDS.

SECTION: PALS-C cloning for saturation mutagenesis
17.4. Perform gel purification using   .

SECTION: PALS-C cloning for saturation mutagenesis
18.1. PALS-C Step 5: Use type2M enzyme   and   to remove templates.

     DpnI      0.5 μL        MspJI      0.5 μL        CutSmart      5 μL        Enzyme activator      1 μL        Purified Step4 product      500 ng        Water      To 50 μL   

     37 °C (Lid: 60 °C)      1 hr        12 °C      Forever   

SECTION: PALS-C cloning for saturation mutagenesis
18.3. PALS-C Step 6: Use Primer F2 and primer R2 to amplify full-length strand.

     Q5 Rxn buffer      10 μL        Q5 Enh buffer      10 μL        10 mM dNTPs      1.5 μL        Purified Step5 product      100 ng        10 μM F2      2.5 μL        10 μM R2      2.5 μL        Q5 polymerase      1 μL        Water      To 50 μL   

     98 °C       hot start        98 °C      5 min        98 °C      6 s        Annealing temperature      20 s        72 °C      Elongation time        Repeat 3-5 for 34 more cycles        72 °C      5 min        12 °C      Forever   

SECTION: PALS-C cloning for saturation mutagenesis
18.2. Perform column purification per manufacturer's instruction.
SECTION: Transduction
11. One day later, refresh medium and add final concentration 8 µg/mL  polybrene.
SECTION: Staining for FFC and FACS
33.1. Remove and discard supernatant.
SECTION: Staining for FFC and FACS
33.2. Resuspend in 3 mL   supplemented in 30 µL  .
SECTION: Staining for FFC and FACS
37.1. Spin down at 700 x g, 10 min, 4 °C. 
SECTION: Staining for FFC and FACS
37.2. Discard supernatant.
SECTION: Staining for FFC and FACS
37.3. Resuspend in 3 mL MAGIC buffer supplemented with 1:50 .

SECTION: Staining for FFC and FACS
39.1. Spin down at 700 x g, 10 min, 4 °C.
SECTION: Staining for FFC and FACS
39.2. Remove supernatant.
SECTION: Staining for FFC and FACS
39.3. Resuspend in 4 mL  
SECTION: Staining for FFC and FACS
39.4. Filter resuspended cells using  .
SECTION: Lentivirus packaging
6.1. Mix 1.5 mL  , 10 µg  , 2 µg   , 9 µg lentiviral plasmid, and 50 µL   .

SECTION: Lentivirus packaging
6.2. Incubate at Room temperature for 15 min.  
SECTION: Lentivirus packaging
6.3. Add mixture dropwise to HEK293T cells.
SECTION: Lentivirus packaging
6.4. Add 3.5 mL   to the 10 cm dish. 
SECTION: Lentivirus packaging
6.5. Incubate at 37 °C for 4320 min in a cell incubator.
SECTION: Lentivirus packaging
7.2. Add 5 mL   to filtered supernatant. 
SECTION: Lentivirus packaging
8.1. Transfer mixture to 50 mL tube. 
SECTION: Lentivirus packaging
8.2. Centrifuge at 1800 x g, 60 min, 4 °C .

SECTION: Lentivirus packaging
7.3. Incubate on a rocker at 4 °C 4320 min.   
SECTION: Lentivirus packaging
8.3. Discard supernatant. 
SECTION: Lentivirus packaging
8.4. Resuspend pellet in 200 µL  .
SECTION: Immunofluorescence
47. Plate MB135 cells
SECTION: Immunofluorescence
47.2. Seed cells into each well.
SECTION: Immunofluorescence
47.4. Differentiate cells for 3-7 days until myotubes are formed.
SECTION: Immunofluorescence
48. Fix cells.
SECTION: Immunofluorescence
48.2. Fix with 4%   for 10 min at Room temperature.
SECTION: Packaging and infection of rVSV/ppVSV
58. Determine 50% tissue culture infectious dose using Spearman-Karber method.
SECTION: Packaging and infection of rVSV/ppVSV
56.1. Seed HEK293T cells in a well of a 6-well plate. Incubate @ 37 °C   .

SECTION: Packaging and infection of rVSV/ppVSV
56.2. Once cells reach 70-90% confluency, transfect cells using 4 µg LASV-GPC plasmid and   per manufacturer's instructions. Incubate @ 37 °C 1440 min .
SECTION: Packaging and infection of rVSV/ppVSV
56.3. Add ppVSVΔG-VSV-G (MOI=0.5). Calculate viral dose given estimated cell number of ~2 M. 


SECTION: PALS-C cloning for saturation mutagenesis
15. Calculate plasmid template input weight:
Coverage = 10^6
Oligo library input weight = (gene cds length * coverage * block number) * (relative moleuclar mass of one oligo)/(Avogadro constant)
Plasmid template input weight = [(oligo library input weight) * (plasmid length) * 2]/(oligo length)

SECTION: Packaging and infection of rVSV/ppVSV
56.5. The next day, collect the newly generated viral particles (referred to as ppVSV-LASV-GPC-Generation1).  Perform titration to determine viral titer.
SECTION: Packaging and infection of rVSV/ppVSV
57.1. Seed 6M HEK293T cells in a 10-cm dish.  Incubate @ 37 °C   .
SECTION: Packaging and infection of rVSV/ppVSV
57.2. Transfect cells using 30 µg LASV-GPC plasmid and   per manufacturer's instructions. Incubate @ 37 °C 1440 min .
SECTION: Packaging and infection of rVSV/ppVSV
57.3. Add ppVSV-LASV-GPC-Generation1 (MOI=0.1) to the well.  Determine viral dose using estimated cell number of ~12M. 

SECTION: Packaging and infection of rVSV/ppVSV
56.4. 60 min later, remove medium, wash with   , and add fresh medium to the well. 
SECTION: Packaging and infection of rVSV/ppVSV
57.4. 60 min later, remove medium, wash with   , and add fresh medium to the well. 
SECTION: Packaging and infection of rVSV/ppVSV
57.5. The next day, collect the newly generated viral particles (referred to as ppVSV-LASV-GPC-Generation1).  Perform titration to determine viral titer.