Apr 04, 2024
Loss of Function Mutagenesis Protocol
- Lilia Peyton1,
- Ada McCarroll1,
- Hanwen Zhang2
- 1Northshore University Healthsystem;
- 2Endeavor Health/Northshore
Protocol Citation: Lilia Peyton, Ada McCarroll, Hanwen Zhang 2024. Loss of Function Mutagenesis Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov19ymklr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 03, 2024
Last Modified: April 04, 2024
Protocol Integer ID: 97721
Abstract
This protocols offers a description of the loss of function mutagenesis procedure for induced pluripotent stem cells.
Materials
- mTeSR Plus:
Catalog # 100-0276,mTeSR‱ Plus cGMP Pluripotent Stem Cell Maintenance Medium | STEMCELL Technologies
- Matrigel
- Geltrex
- DPBS
- ReLeSR
- Cryovials
- Barcoded Cryovials
- Accutase
- Plasmids
- Opti-MEM
- Polyamine Supplement: 7739/1, https://www.rndsystems.com/products/polyamine-supplement-x1000-lyophilized_7739
- 5mL corning round bottom tubes with blue strainer cap
- 5mL corning round bottom sample collection tubes
- DNA Quick Extract: NC9904870, https://www.fishersci.com/shop/products/quick-extract-dna-extrac-50ml/NC9904870#?keyword=NC9904870
F and R Primers
Taq Polymerase
- SAP+ SAP Buffer: 78-390-5000UN, https://www.fishersci.com/shop/products/shrimp-alkaline-phosphatase-sap-4/783905000UN
- Exonuclease: 70-073-X5000U, https://www.fishersci.com/shop/products/exonuclease-i-standard-concentration-10-units-l-2/70073X5000U
- Recombinant Anti-Oct4 antibody: ab181557, https://www.abcam.com/products/primary-antibodies/oct4-antibody-epr17929-chip-grade-ab181557.html
- Anti-SSEA4 antibody: ab16287, https://www.abcam.com/products/primary-antibodies/ssea4-antibody-mc813-70-ab16287.html
- Donkey anti-Goat IgG (H+L) Cross Adsorbed Secondary Antibody, Alexa Fluor 568:
A11057
- Donkey anti-Chicken IgY (H+L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor 647:
A78952
- Donkey anti-Rabbit IgG (H+L) Cross Adsorbed Secondary Antibody, DyLight 755:
PISA510043
- Thermo Scientific DAPI Solution (1mg/mL):
EN62248
iPSC Maintenance
iPSC Maintenance
Change media every other day – mTeSR Plus with 1ml primocin
Always warm media to room temperature before use
Aspirate using glass pipette attached to vacuum and replenish with mTeSR plus
Passaging
Passaging
Passage every 4-6 days (whenever colonies are 70-80% confluent- depends on the density that you plate)
Coat desired number and size of plates with matrigel/geltrex and incubate overnight or >4hr at 37C
On day of passage, cool the coated plates at room temperature for >1hr then aspirate matrigel/geltrex then add appropriate amount of media for well size
Aspirate media from cells and wash with DBPS
Add appropriate amount of ReLeSR and aspirate within 1 minute
Incubate at RT for 4-5 minutes
Spray wells with media (1ml/well for 6w) with p1000 then hold the plate in one hand and use other hand to firmly tap the side of the plate to detach the cells
Use p1000 to wash/collect the detached cells- gently pipet the cells 2-4 times, being careful not to break the aggregates into single cells and transfer to labeled 15ml tube
Plate the cell aggregate mixture at the desired density onto the pre-coated wells containing media
Usually 1:10 - 1:20 or 1:4-1:5 depending on cell density
Check that there is an appropriate number of cells under the microscope then place in the incubator at 37C; Move the plate in several quick, short, back-and-forth and side-to-side motions to evenly distribute the cells
Do not disturb the plate for 24 hours
Thawing Cells
Thawing Cells
Prepare coated 6w plates with 1 well for each cryovial (same as step 1 above)
Can prepare 2 wells for each cryovial – depending on how confluent you want cells to be thawed
On day of thawing, cool plates at RT >1hr before use but do not add media
Label 15ml tubes for each cryovial that is being thawed
Take cryovials out of liquid nitrogen
Thaw cryovials in dry bath until only one ice crystal is left
Use a glass pipette to remove cells from cryovial and transfer to labeled 15ml tube
Slowly add 5ml mTeSR Plus dropwise into 15ml tube to avoid shocking the cells – rotate the tube while adding media to ensure even distribution
Centrifuge 3 mins at 300g
Aspirate supernatant – okay to leave a little film to avoid disturbing the pellet
10. Aspirate coating matrix from prepared plate
Use glass pipette to add 2ml mTeSR Plus with 5µM ROCK-Inhibitor and forcefully mix to break up pellet
Add 2ml/well cell suspension to coated plate
Incubate at 37C and change media with regular mTeSR Plus the next day/24 hours after to remove ROCK-I
Freezing Cells
Freezing Cells
Determine which wells on 6w plate are ready to be cryopreserved
Prepare labels and vials (2 vials per clone) – use label machine to create labels, ex. “g1b1c1A1”
Put label on vial and scan barcode on bottom of vial in excel to connect it to the right clone
Aspirate media from wells and wash with DPBS
Add 1ml ReLeSR per well and aspirate within 1 minute
Incubate at RT for 4-5 minutes
Spray wells with media (1ml/well for 6 well) then hold the plate in one hand and use other hand to firmly tap the side of the plate to detach cells
Use P1000 to wash/collect the detached cells – gently pipet the cells 3-4 times, being careful not to break cell aggregates into single cells – and transfer to labeled 15 ml tube
Centrifuge for 3 min at 300g
Gently aspirate the supernatant without disturbing the pellet
Use 5ml glass pipette to resuspend pellet in 2 ml mFreSR
10. Transfer 1ml of cell aggregate mixture into each labeled cryovial
11. Transfer cryovials into isopropanol container then put it in -80C freezer for 48 hrs
12. Transfer cryovials from isopropanol containers to boxes from LN2 on dry ice
Need three people – one checks which box the vial should go in, one scans it into the excel spreadsheet, one physically moves vial from isopropanol container to box on dry ice; Transfer all vials at same time to ensure correct placement and ease for future shipment
Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
Prepare Cells for Transfection *all procedures in media with 5uM ROCK-I*
Cell are ready to be plated for transfection when they are 70-80% confluent. Use 3-5 wells from a 6w plate depending on confluency
Coat 2-24w plates 1 day before experiment – incubate at 37C overnight
Plate two different densities and determine which to use on day of transfection based on confluency and cell survival
On day of experiment, cool plates >1hr and set out appropriate amounts of media and accutase
Aspirate coating matrix and add 0.5ml media to each well – return plates to incubator until ready to use
Aspirate media and wash cells with DPBS
Add 1ml/well accutase and incubate at 37C for 5 min; if cells are not easily detaching incubate 7-10 mins
While incubating, add 2ml media to 15ml tube
Following incubation, gently pipet cells twice and transfer to the prepared 15ml tube
Centrifuge 300g for 3 min
Aspirate supernatant – okay to leave a little film to not disturb pellet
Resuspend the pellet with a glass pipette in 4ml media – take 10µl for counting
Count cells using hemocytometer or countess machine
Calculate 2e5 and 1.5e5 cells per well
Resuspend then aliquot with glass pipette for 25 wells into a new 50ml tube; repeat for both densities
Centrifuge 300g for 3 min
Remove plates from incubator and label for each condition
Aspirate supernatant – okay to leave a little film to not disturb pellet
Resuspend cells in 12.5ml media
Add 0.5ml cell suspension for each well in 24w plate
After every 6 wells, move the plate in several quick, short, back-and-forth and side-to-side motions to evenly distribute the cells
Incubate at 37C overnight
Repeat steps 17-21 for other density
Transfection for iPSCs in 24w Plate
Transfection for iPSCs in 24w Plate
Before transfection, decide which density has better cell survival and density
Change media for selected plate with antibiotics free mTeSR Plus with 5µM ROCK-I (700ul per well)
Prepare working plasmid solution using endo-free TE buffer in hood:
pEF-AncBE4max (from Brafman): 500ng/µl
pEF-BFP: 200ng/µl
pDT-sgRNA (with variant specific gRNA cloned): 300 ng/µl
Prepare tubes for each sgRNA, one for DNA master mix, and one for Lipo master mix (26 tubes)
Prepare DNA MM:
625ul Opti-MEM
37.5ul AncBE4max
37.5ul BFP
Aliquot 28ul of DNA MM into each sgRNA tube
Add 1µl of each sgRNA to their corresponding tubes containing DNA MM
Prepare Lipo Master mix:
625ul Opti-MEM
82.5ul Lipofectamine STEM
Aliquot 28.3ul of LIPO MM to each sg RNA tube and mix well
Incubate at RT for 10 min
Towards end of incubation, label prepared 24w plate with corresponding gene numbers
Add 50µl complex dropwise into the correct well and gently swirl plate to ensure even distribution
Return plate to incubator
24 hours post transfection, refresh media with regular mTeSR Plus
48 hours post transfection, refresh media with regular mTeSR Plus
48 hours post transfection, coat 24 – 96w plates for sorting
72 hours post transfection, prepare for FACS
Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
Prepare for FACS *all procedures use mTeSR plus with CEPT cocktail*
Check 24w plate with confocal microscope before preparing for FACS – determine that there is enough GFP+ cells to sort
Set out media and accutase to warm to RT – add CEPT components (1:10,000 chroman I, emricasan, and transISRIB; 1:1,000 polyamine supplement) to media then filter before use
Cool 96w plates >1hr at RT then aspirate coating matrix and add 120µl media. Return plates to incubator for later use.
Complete following procedure row by row (6 wells) for 4 batches of 6 wells.
Wash with DPBS
Add 300µl accutase to each well
Incubate at 37C for 5 min; incubate longer 7-10 mins if needed
During incubation, label 15ml tubes for each condition and add 1 ml of media to each tube
Following incubation, gently pipet cells once and transfer to corresponding 15ml tubes
Centrifuge 300g for 3 min
Aspirate supernatant – careful not to disturb pellet
Resuspend pellet in 700µl media; 500ul if low cell confluence and 1ml if cell confluence is high
Filter cell suspension twice using two different 5ml corning round bottom tubes with blue strainer cap
Replace the blue cap with a filterless white cap from a sample collection tube to avoid contamination
Put samples on ice immediately
Sorting with FACS Aria – 1 double positive cell (BFP + GFP) into one well on 96w plate
After sorting, centrifuge the plates at 300g for 1 min to help cells attach
Return plates to incubator ASAP
Repeat until all 4 batches are done
Care after Sorting *All media changes with regular mTeSR Plus to dilute CEPT cocktail)
Care after Sorting *All media changes with regular mTeSR Plus to dilute CEPT cocktail)
24hr post sorting: do not disturb cells
48hr post sorting: use robot pipettor to add 50µl media
72hr post sorting: use robot pipettor to change 50µl media
96hr (Day 4) post sorting: use robot pipettor to change 120µl media
144hr (Day 6) post sorting: aspirate 120µl, add 100µl media
Afterwards, use robot pipettor to change 100µl media every other day
It takes 10-14 days for a single cell to grow into a colony with appropriate size for picking Approximately one week post sorting, determine which wells have colonies and start to mark/plan for colony picking
Colony Picking
Colony Picking
Day before experiment: Determine which plates you will be picking from based on size and colony morphology – mark 12 best colonies with microscope; Each colony marked plate will go onto one row on two identical 96w plates (see example below) – one plate for maintaining cells and one plate for DNA extraction
Day before experiment: Coat one row on 96w plate for each plate that is ready to be picked- normally 2-3 plates for the first round of picking depending on growth rate, then another 2 plates for the second round
Ex. 8 marked plates = one full 96 well plate
On day of the experiment, cool plates >1hr RT then aspirate coating matrix and add 80µl mTeSR Plus with 5µM ROCK-I to each well (maintenance plates); an uncoated empty 96w plate copy is needed for each coated plate for DNA extraction (DNA plates). Label plates to correspond to the correct genes and indicate whether they are the "DNA plate" or the "maintenance plate"
Pick colonies from one marked plate at a time following the below procedures
Aspirate 50-70µl media from marked wells so around 70-100µl left
Set p200 pipettor at 50µl and scrape in marked area of well ~30 seconds then collect as many aggregates as possible in 50µl volume
Transfer the 50µl aggregates into a well on the maintenance plate, mix well by pipetting 3 times
Withdraw 50ul cell suspension from maintenance plate and add to DNA plate
Repeat well by well until row is completed
Repeat process until maintenance and DNA plates have all intended genes
Centrifuge maintenance and DNA plates at 300g for 3 min
Return maintenance plate to incubator
Quickly aspirate media without disturbing the cells from the DNA plate
Plate can be stored at -20C temporarily, no more than an hour – while picking other maintenance and DNA plate
Add 16µl DNA quick extract to well on DNA plate and gently scrape/flush the well bottom to collect all cell aggregates – one well at a time, on ice
Transfer 16µl solution into a 96w PCR plate – keeping the same format as the DNA plate
Briefly spin PCR plate
Run DNA extraction protocol on thermos cycler (1st step in Sanger sequencing workflow)
Follow Sanger sequencing protocol to get results in 2-3 days
Sanger Sequencing
Sanger Sequencing
DNA Extraction -> Amplification PCR -> SAP -> Sequence PCR -> Purification -> Sequencing
DNA Extraction (cell lysate in 16ul solution)
DNA Extraction (cell lysate in 16ul solution)
Run DNA extraction protocol on thermocycler:
a. 65C for 15 min
b. 68C for 15 min
c. 95C for 10 min
d. 4C for ever
Go directly to PCR Amp step, do not pause here!
PCR Amplification
PCR Amplification
Prepare working primer from stock solution (100uM to 10uM)
Prepare master mix for each gene and for appropriate number of wells, accounting for 20% extra:
AmpPCR mix 1x (ul)
Water 5.5
10X PCR Amplification Buffer 1
10X PCR Enhancer 1
50mM MgSO4 0.3
10mM dNTP 0.2
Primer F 0.2
Primer R 0.2
Polymerase 0.1
Transfer prepared AmpPCR mixes into corresponding rows on a 96w PCR plate (8.5ul per well)
Add 1.5ul quick extracted DNA to plates and mix 3 times
Seal plates and briefly centrifuge 2000g ~5 seconds
Run PCR Amp protocol on thermocycler:
a. 95C for 10 min
b. 95C for 30 sec
c. 53C for 1 min 30 sec
d. 72C for 1 min
e. Step b-d 40 times
f. 72C for 10 min
g. 4C for ever
Directly to SAP step or can pause here; leave plate at 4C <18 hrs
SAP Reaction (5ul mix + 5ul PCR product= 10ul total)
SAP Reaction (5ul mix + 5ul PCR product= 10ul total)
Prepare master mix:
SAP mix 1x(ul)
Water 3.9
10x SAP buffer 0.5
SAP 0.5
E. coli exonuclease I 0.1
Pipette 5ul SAP mix into each well on a 96w plate
Add 5ul PCR product to each well on SAP plate, pipetting 3 times to mix
Briefly centrifuge 2000g ~5 sec
Run SAP protocol on thermocycler:
a. 37C for 50 min
b. 95C for 15 min
c. 4C for ever
May pause here, leave the plate at 4C <18hr, or go directly to Seq PCR step
Seq PCR (5ul mix + 5ul SAP product = 10ul total)
Seq PCR (5ul mix + 5ul SAP product = 10ul total)
Prepare master mix following table above for each gene - only use either F primer or R primer, do not use both! Be aware of primer records, some genes require more of less big dye
Seq PCR mix 1x (ul)
Water 2
Big dye 2
Primer F or R
Pipette 5ul mix into a 96w PCR plate, being careful to be consistent with gene placement
Add 5ul SAP product, pipetting 3 times to mix
Seal the plate and briefly centrifuge 2000g ~5 sec
Run Seq PCR protocol on thermocycler:
a. 96C for 1m in
b. 96C for 10 sec
c. 55C for 5 sec
d. 60C for 4 min
e. Step b-d for 25 times
f. 4C for ever
May pause here, leave the plate at 4C <18 hrs or go directly to purification step
Purification
Purification
Prepare fresh 75% isopropanol
Add 40µl 75% isopropanol into each well, pipetting 3 times to mix
Gently attach a sealing film and incubate at RT for 20 min
Centrifuge plate at 2700g for 30 min
Gently invert the plate onto paper towel
Add 150µl 75% isopropanol in each well, do not mix!
Reseal the plate and centrifuge at 2700g for 12 min
Gently invert the plate onto a paper towel
Keep the plate inverted and transfer to a new paper towel and centrifuge at 200g for 1 min to remove residual isopropanol
11. Directly go to next step
Prepare for Loading
Prepare for Loading
Add 16µl HiDi formamide to each well, pipetting 3 times to mix
Seal the plate and centrifuge at 2000g for ~5 sec
Run the forma protocol on thermocycler:
a. 95C for 3 min
b. 4C for 2 min
c. 4C for ever
Centrifuge at 2000g for 1 min
Ready to load
Setup 3730 for Sequencing
Setup 3730 for Sequencing
Start 3730 software
Select instrument status
Connect the polymer bottle – set waste bottle and cap on white tray
Run bubble remove wizard twice (check for bubbles) then fill array
In plate manager – import or build a new template for today’s run
Go to run scheduler – search the plate ID and add plate in the queue
Make sure to remove any sealing film on PCR plate (will damage array if not)
Put the PCR plate in the black bottom, add a clean grey septa on it, then attach the white top – make sure it clicks closed and have angled corner of black bottom on top right of PCR plate, A12 on PCR plate goes in angled corner to have correct orientation
10. Put the PCR plate sandwich into the “In stack” drawer – make sure to close the door properly
11. When the system detects plates in the “In stack”, click “play” button – top left in the software – then click “yes” to confirm and start the program
12. Takes ~2 hours to run a full 96w plate
13. After run, copy data to a flashdrive
14. Exit the software, shutdown the computer, and return the polymer to 4C
Analyze data on computer with SeqScape
18. Check each sample for expected editing and record – use to decide which wells to expand
Expand Selected Clones
Expand Selected Clones
After viewing Sanger sequencing results, label 4 wells to keep on 96w plate – two to expand and two as backups; prioritize homozygous clones and clones with the best morphology
24hr post picking, add 50µl regular mTeSR Plus into selected wells
48hr post picking, change 80µl media for selected wells
Change 100µl media every other day
Once colonies have grown to proper size (typically 4-6 days), expand from 96w to 6w
Coat one well on 6w plate for each well on 96w plate that is ready to be expanded – incubate >4hr
Cool 6w plates >1hr at RT and label for each well being expanded – cell line, gene #, clone, gene name, passage #
Aspirate coating matrix and add 2ml media to each well
Then aspirate media from wells ready to be expanded with p200
Add 100µl ReLeSR to wells and immediately aspirate – add and aspirate from wells one by one
Incubate for 3 min at RT
Add 100µl media to each well
Scrape colonies one by one using p200 – scape entire area
Aspirate full volume from well and add to corresponding well on the 6w plate – move back and forth, right and left to gently disperse cells
Passage cells once to have 2 wells on 6w plates with appropriate confluence for each clone – one well is needed for cryopreservation and one well for RNA isolation
RNA isolation
RNA isolation
Add 800ul buffer RLT Plus to expanded 6w plates with 70-80% confluence
Pipet 30-60 seconds, washing the well until the solution is homogenized and clear
Add 350ul each to two locking lid 1.5 ml microcentrifuge tubes
Store in -80C for temporary storage. Within a week, isolate RNA using the RNeasy Plus Mini Kit
Transfer cell lysate to QIA shredder and centrifuge 2 min at max speed
Transfer liquid to gDNA elimination column and centrifuge 15 sec at 11,000 rpm
Discard flow through and keep spin column and tube
Add 350ul 70% ethanol to lysate and mix until homogenized
Transfer to RNA easy spin column and centrifuge 15 sec at 11,000 rpm
Discard flow through and keep spin column and tube
Add 700ul buffer RW1 to RNA easy spin column and centrifuge for 1 min at full speed
Discard flow through and keep spin column and tube
Add 500ul buffer RPE to RNA easy spin column and centrifuge 15 sec at 11,000 rpm
Discard flow through and keep spin column and tube
Repeat previous step, centrifuge for 2 min rather than 15 sec
Transfer RNA easy spin column to collection tube and centrifuge 1 min at full speed
Transfer RNA easy spin column in labeled 1.5l tube and add 35ul RNAse free water directly to the membrane
Centrifuge for 1 min at 11,000 rpm
Discard RNA easy spin column and place 1.5ml tube directly on ice
Measure the concentrations of each sample using the NanoDrop
Aliquot 20-25ul into new labeled 1.5ml tubes and wrap well with parafilm to ship to Novogene
Immunohistochemistry: Staining for Quality Control of iPSCs
Immunohistochemistry: Staining for Quality Control of iPSCs
Fixation
Coat coverslips the day before in 4w dish
Start from iPSCs culture on 96w plate- plate on 12mm 1.5 image-grade glass
Prepare 4% PFA solution in 1xPBS (16% stock in PBS, 400ul per well)
Aspirate old media and add 4% PFA into each well
Incubate at RT for 12-15 min
Aspirate PFA, wash cells 3 times with PBS
After third wash, add 1ml PBS + 0.03% sodium azide to each well
Wrap the 4w dish using parafilm and store the plate in 4C; you can pause her for a few days
Permeabilization
Prepare blocking buffer (3% BSA in 0.1% PBST)
Prepare 1% triton X-100 in PBS
Add 500ul PBST to each well and incubate at RT for 15 min
Blocking
Aspirate PBST from permeabilization step
Add 1ml blocking buffer to each well (3% BSA in 0.1% PBST (0.1% triton in 1x PBS))
Incubate RT for 1 hr; can leave in 4C overnight
Primary Antibody
Following vendors instructions, prepare antibodies in blocking buffer; can use 1ug/ml to start if no instructions are available
Aspirate blocking buffer
Add 350ul primary antibodies in blocking buffer to each well and incubate RT for 1.5 hr; can leave overnight if necessary but increase the volume of solution
Wash
Aspirate primary antibody
Add 1ml PBS or 0.1% PBST to each well - 3 times, incubating for 5 min at RT each time
Secondary Antibody
Prepare 1:1000 dilution in blocking buffer; make sure to se different hosts and colors and match the primary antibody
Aspirate wash
Add 350ul secondary antibody in blocking buffer to each well and incubate at RT for 1 hr. It is light sensitive at this point so be sure to incubate in the dark (cover in foil, keep in a drawer or cabinet)
Wash
Repeat same washs step as above but incubate in the dark
DAPI
Ready to use solution in 4C fridge (0.5ug/ml in PBS)
Aspirate wash
Add 400ul DAPI solution to each well and incubate at RT for 10 min
Wash
Aspirate DAPI
Briefly wash each well with 1ml PBS
Thaw diamond anti-fade solution
Mount
Add one drop of diamond anti-fade solution to slide
Aspirate PBS and use tweezers to grab and invert the coverslip to make sure the cells contact the anti-fade reagent
Slides can be stored in the dark, > overnight to let the anti-fade dry
Ready for imaging