Loss of Function Mutagenesis Protocol

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

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

Do not disturb the plate for 24 hours

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

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

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

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

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

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

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

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

DNA Extraction -> Amplification PCR -> SAP -> Sequence PCR -> Purification -> Sequencing

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!

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

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

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

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

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

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

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

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

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