TITLE: Generation of A549 PPM1H BromoTag CRISPR/CAS9 knock-in cell line
AUTHORS: Tran Le Cong Huyen Bao Phan, Thomas Macartney, Dario Alessi

[DESCRIPTION]
This protocol details the generation of A549 PPM1H BromoTag CRISPR/CAS9 knock-in cell line.


[STEPS]
SECTION: Transfection for PPM1H BromoTag
1. 
 
Seed wild type A549 cells approximately 60 – 70% confluency in 3 mL media/well (DMEM + 10% FBS (Foetal Bovine Serum) + 1% penicillin/streptomycin + 1% L-glutamine) in 6-well plates. Preferably plate 2 wells per guide + donor DNA (one well for cell growing and one for immunoblotting). 
 

SECTION: Transfection for PPM1H BromoTag
2. After 16 – 20 hours of cell seeding, perform transfection using LipofectamineLTX. 
 

SECTION: Transfection for PPM1H BromoTag
2.1. If use only one guide, then use at a ratio 1:1 (guide 0.5 µg : donor DNA 0.5 µg).

DNA : LipofectamineLTX ratio = 1 µg : 3.5 µL
DNA : PLUS Reagent ratio = 1 µg : 1 µL
Transfection (per well):

Mix A: 
 

Mix B:  
 

SECTION: Transfection for PPM1H BromoTag
2.2. Add mix A to mix B, incubate at Room temperature for 5 min, add gently 300 µL mix dropwise to cells.
SECTION: Transfection for PPM1H BromoTag
3. After 24 hours of transfection, aspirate old media and add 3 mL fresh media/well containing puromycin at 3 µg final concentration. Treat cells for 2 days before adding fresh media. 
 

SECTION: Transfection for PPM1H BromoTag
4. Allow cells to grow until confluent. Spit cells from 6-well plates into 10 cm petri dishes, then let cells grow until confluent. Cells from 1 dish are stored in freeze media (10% DMSO + 90% FBS) at -80 °C for single cell sorting later, cells from the other dish are used for immunoprecipitation to confirm the presence of PPM1H BromoTag.
SECTION: Immunoprecipitation of PPM1H from the pools
5. Aspirate media from 10cm dish, wash cells twice with PBS. Add 300 µL lysis buffer into the dish and scrape it using a suitable scrapper. Transfer the lysate into a 1.5ml Eppendorf tube.
SECTION: Immunoprecipitation of PPM1H from the pools
6. Centrifuge the lysates at 17000 x g, 10 min, 4 °C. Transfer supernatant into a new 1.5ml Eppendorf tube.
SECTION: Immunoprecipitation of PPM1H from the pools
7. Quantify protein concentration using Bradford assay.
SECTION: Immunoprecipitation of PPM1H from the pools
8. Immuno-precipitate PPM1H using anti-PPM1H coupled beads. 500 µg of lysates is incubated with 10 µL of beads for 120 min in the cold room. 
 

SECTION: Immunoprecipitation of PPM1H from the pools
9. Wash the beads 3 times with PBS, then add 20 µL of 2X SDS-PAGE loading buffer, incubate for 10 min at 70 °C.
SECTION: Immunoprecipitation of PPM1H from the pools
10. Collect the supernatant by centrifuging through a   Spin-X Centrifuge tube filters.

SECTION: Immunoprecipitation of PPM1H from the pools
11. Add beta-mercaptoethanol to the eluate to a final concentration 1%.
SECTION: Immunoprecipitation of PPM1H from the pools
12. Boil the eluate at 95 °C for 5 min, then subjected to immunoblot analysis.
 

SECTION: Single cell sorting
13. After confirming the presence of PPM1H BromoTag in transfected cells, re-grow cells stored at -80 °C from the section Transfection for PPM1H BromoTag in 10 cm plate in 10 mL complete media.
 
 Complete media
      DMEM    10%FBS     1% penicillin/streptomycin     1% L-glutamine  

SECTION: Single cell sorting
14. One day before cell sorting: add 200 µL media (DMEM + 20%FBS + 1% penicillin/streptomycin + 1% L-glutamine) into a 96-well plate, keep in 37 °C incubator supplemented with 5% CO2.
SECTION: Single cell sorting
15. On the day of cell sorting:
SECTION: Single cell sorting
15.1. Trypsinise cells (3 mL/dish), stop trypsinisation with 10 mL media (DMEM + 10%FBS + 1% penicillin/streptomycin + 1% L-glutamine), transfer to a 15ml falcon tube. 
SECTION: Single cell sorting
15.2. Spin down at 250 x g, 3 min.

SECTION: Single cell sorting
15.3. Remove media, resuspend cells in 2 mL of media (DMEM + 1%FBS + 1% penicillin/streptomycin + 1% L-glutamine).
SECTION: Single cell sorting
15.4. Sort single cells with positive GFP signal into a 96-well plate prepared one day before. 
 

SECTION: Single cell sorting
15.5. After finishing cell sorting, spin down the 96-well plate at 250 x g, 1 min to ensure cells are at the bottom of the plate.
SECTION: Single cell sorting
15.6. Leave cells to grow for approximately 2 weeks in 37 °C incubator supplemented with 5% CO2.

SECTION: Growing clones
16. Check every clone under microscopy. After approximately 2 weeks, a single cell will grow into a colony.
SECTION: Growing clones
17. Split growing cells from single clones into 6-well plates (2 mL media/well, DMEM + 10%FBS + 1% penicillin/streptomycin + 1% L-glutamine). Let them grow until confluent.
SECTION: Growing clones
18. Split cells from single clones into 2 wells of 6-well plates, wait until they are confluent. 
SECTION: Growing clones
19. Lyse cells from one well for screening (section Screening positive clones), and store cells from another well in freeze media (10% DMSO + 90% FBS) at -80 °C for short term storage. 
 

SECTION: Screening positive clones
20. Lyse cells from individual clones recovered from cell sorting with 150 µL of lysis buffer.
SECTION: Screening positive clones
21. Analyse the lysates by immunoblotting with anti-PPM1H or anti-BromoTag antibodies (Fig1C). Wild type cells used as a control for wild type PPM1H with molecular weight approximately 56 kDa. PPM1H BromoTag protein runs at a size around 71 kDa.
SECTION: Screening positive clones
22. Select clones not showing a band of wild type PPM1H for further analysis. To confirm the presence of PPM1H BromoTag in these clones.
SECTION: Screening positive clones
22.1. Seed A549 wild type and knock-in cells at ~ 70% confluency in 6-well plates 1 min.

SECTION: Screening positive clones
22.2. Treat cells with 300 nanomolar (nM) AGB1 or 300 nanomolar (nM) cis-AGB1 (epimer inactive degron) compounds for 240 min.
SECTION: Screening positive clones
22.3. Lyse cells, then analyse by immunoblotting with anti-PPM1H antibody. 
 

SECTION: DNA sequence to characterise homozygous A549 knock-in PPM1H BromoTag cell line
23. Extract genomic DNA from several positive clones using Qiangen kit according to the manufacturer’s instructions.
SECTION: DNA sequence to characterise homozygous A549 knock-in PPM1H BromoTag cell line
24. Perform PCR to confirm the presence of PPM1H BromoTag in potential homozygous clones (Fig1D).

A pair of primers (Cter 5F2: 5’- CTTGCTGAACTTACATTGGTCAAGAGG and BromoTag R: 5’-ACTTGATTGTGCTCATGTCCATGG) are used to amplify an amplicon with expected size at 1062 bp (Fig1A-B).
Water and wild-type (WT) A549 should be included as negative controls.
KOD Hot Start Polymerase kit is used as described below.
 
SECTION: PCR reaction setup
25.  
  ReagentsVolume (μl)10X buffer2dNTP (2mM each)225mM MgSO41.2DMSO1.210 μM forward primer0.610 μM reverse primer0.6Genomic DNA (75ng)1KOD polymerase0.4Nuclease-free water11Total volume20

SECTION: Cycling conditions
26. 
   StepTime98°C2 min98°C10 s35 cycles60°C20 s70°C20 s/kb70°C5 min4°Chold

SECTION: Cycling conditions
27. Select clones showing a band at expected size (1062bp) for further analysis (Fig1D). Clone PCR products from the positive clones into the StrataClone PCR cloning vector pSC-B-amp/kan according to the manufacturer’s instructions (Stratagene).
SECTION: Cycling conditions
28. After overnight incubation at 37 °C, pick randomly 12-20 bacterial white colonies for plasmid DNA analysis and grow in 3 mL LB amp media 240 min. 
 

SECTION: Cycling conditions
29. Prepare miniprep DNA plasmids from the selected colonies according to the manufacturer’s instructions (Qiagen). 
SECTION: Cycling conditions
30. Identify plasmids containing the PCR product insert (1062bp) by restriction analysis with EcoRI digestion according to the manufacturer’s instructions (ThermoFisher Scientific) (Fig1E).
SECTION: Cycling conditions
31. Send all the miniprep plasmids for DNA sequence with M13 forward primer (5’- GTAAAACGACGGCCAGTG) and M13 reverse primer (5’- GGAAACAGCTATGACCATG) to identify homozygous knock-in clones.
SECTION: Cycling conditions
32. Store homozygous A549 PPM1H BromoTag clones in liquid nitrogen for long-term storage.