Alice:Single cell tracing and RNA-seq Lab Notes/Brainbow Protocols

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BAC mediated Brainbow knock-in at the HPRT locus[edit]

Cloning and cell culture general protocols[edit]

plasmid transformation:
TOP10 chemical competent cell (50ul) + plasmind (0.5ul)
incubate on ice for 30 min
heat shock at 42C for 1min
quickly transfer onto ice and incubate for 2 min
add 1ml LB and shake in 37C incubator for 1 hour
evenly spread onto LB plate and incubate in 37C incubator overnight (~15-20 hours)
NEXT DAY, one should notice small satellite colonies surrounding the big one, which it is most likely the correct clone (able to grow strongly on antibiotic plate)
prepare 3ml LB media in 10-12ml culture tube
use clean (autoclaved) pipette tip to pick up single colony and drop into (or rinse) into the 3ml LB tube
then shake at 37C overnight (always the best to start in late afternoon)
(leave the plate in 4C if not enough time to pick single colony at the time)
NEXT DAY, prepare glycerol stock of the overnight culture:
add 400 ul of 30% autoclaved glycerol, and 400 ul of overnight culture (final concentration of glycerol should be around 15%)

BAC Competent cell construction and transformation[edit]

  1. Take SW102 (derived from DY380 strain and therefore contains the λ prophage recombineering system) glycerol stock, spread on plate using pipette tips, incubate at 30C overnight
  2. Pick a single colony and shake in 3 ml low-salt LB overnight (~12-15 hours)
  3. Take out 500 ul from the overnight culture and shake in 25 ml NO-salt LB (sometimes low-salt LB can be used instead, because Na+ can possibly help increase membrane fluidity and conductivity for electroporation)
  4. After ~3 hours, check the OD of the culture, and optimal OD is 0.55-0.6 (use fixed wavelength of 600 nm and visible light, make sure to turn off UV light)
  5. Transfer all the cultured media to 50 ml conical tube
  6. OPTIONAL: if need to do recombineering, then put the culture at 42C for exactly 15 min then quickly transfer to ice (~0C), otherwise can skip it the step during initial prep
  7. After cooling in ice/water mix briefly, centrifuge at 3700 rpm for 5 min
  8. Carefully remove supernatant and add 1 ml of DI H2O (autoclaved and 0.2um filtered clean H2O without any electrolyte, because that will decrease electroporation efficiency)
  9. Re-suspend the pellet by shaking the tube up and down in the ice/water mix
  10. After the pellet has completely resuspended, add 24 ml ice cold DI H2O
  11. Centrifuge at 4500 rpm for 5 min
  12. Repeat the re-suspension step and add 24 ml of DI H2O
  13. Centrifuge at 4700 rpm for 7 min
  14. Carefully remove the supernatant, use kimwipe to wipe off the liquids on the side of the tube (without touching the pellet)
  15. Re-suspend the pellet in the remaining liquid (~50 ul) since it is not possible to remove all the liquid
  16. Use fresh made competent cells to do transformation and also freeze back remaining amount of competent
  17. OPTIONAL: Quickly freeze the competent cells in 25 ul stock using liquid nitrogen (storage time is up to 2 months)
  18. Thaw out the BAC plasmid on ice and transfer 4ul per reaction to a new 1.5ml tube, then add in 25ul of fresh or thawed competent cells prepared previously
  19. Gently flick the tube or pipette slowly to mix well, then transfer the mixture into 0.1 cm electrode Gene Pulser cuvette (dark brown cap)
  20. Incubate the cuvette on ice for few minutes before and after transferred the BAC + competent cell mixture to keep everything cold
  21. Wipe off any liquid on both magnetic sides of the cuvette, check to make sure no bubbles in the sample (which would cause electric spark and fry the samples, if that happens, need to redo electroporation)
  22. Use Bio-rad Gene Pulser II to do electroporation with the setting: 1.75 kV, 200 ohms, 25uF. Press both red bottom at the same time to activate the electrode and wait for the beep to release (pulse time constant reading will be displayed on screen)
  23. Add 900ul low or NO salt LB to resuspend the mixture, and transfer to new 1.5 ml tube and shake in 30C for 50 minutes
  24. Spin at 4000 rpm in bench top centrifuge for 4-5 minutes, then take away the supernatant, plate the palette and small amount of liquid onto LB agar plates overnight in 30C
    1. NOTE: sometimes, low salt LB broth can help with electroporation by increasing the fluidity of cell membrane, and also decrease the conductivity of ions floating in the solution

Plasmid Transformation[edit]

  1. Incubate the competent cells (50 ul) and ligation product (5 ul) on ice for 30 min
  2. Incubate the mixture at 42C for 1 min
  3. Quickly transfer to ice and incubate for 2 min
  4. Add 900 ul LB to recover the competent cells at 37C for 30-45 min
  5. Centrifuge at 4000 rmp for 4 min
  6. Carefully remove the supernatant and then plate the pellet on LB agar plates overnight in 37C incubator

Experimental design and results[edit]

HPRT-Brainbow BAC construction (8/8/2013 – 12/19/2013)[edit]

Brainbow2.1R construct modified with F5 instead of Frt, renamed with BBF5
-	Starting plasmid: BB construct given by Blue, BssHII-BB2.1R-HindIII-XhoI
-	Design F5 oligo (to change frt in brainbow to its variant form F5; refer to Schlake Bode 1994):
forward: 5' cgcgcgaagttcctattcTTCAAAAGgtataggaacttcactagta
reverse: 5' agcttactagtgaagttcctatacCTTTTGAAgaataggaacttcg
forward strand: 5' cgcgc gaagttcctattc TTCAAAAG gtataggaacttc actagt a     3'
forward strand: 5' BssHII (invert repeat, F5 spacer, invert repeat) SpeI HindIII
reverse strand: 3'     g cttcaaggataag AAGTTTTC catatccttgaag tgatca ttcga 5'
reverse strand: 5' HindIII SpeI F5 BssHII
-	Anneal the forward and reverse oligos designed above
-	Use restriction enzyme BssHII and HindIII on BB2.1R plasmid 
       (to cut frt fragment from BB construct, and also introduce F5 fragment and SpeI restriction site) and also the F5 oligo
-	Ligate the F5 oligos to BB2.1R construct
-	Final product: BB-F5 variant-SpeI
CAG-BBF5 construction
-	Starting plasmid: BBF5 from above step (BB construct: SnaBI-NheI-BB)
-	Amplify CAG promoter from another plasmid and introduce several restriction sites next the promoter
-	Primer to amplify CAG promoter:
Forward (EcorV-EcorI-CAG-F): 
GACGGATATCGAATTCTCGACATTGATTATTGA
Reverse (R-CAG-NheI): 
TCAGCGCTAGCCTGCAAATTCTTTGCCAAAATG
-	PCR product of CAG promoter: EcorV (blunt end)-EcorI-CAG-NheI
-	Digest the CAG promoter with EcorV and NheI
-	Restriction enzymes EcorV and SnaBI both creat blunt end product, therefore the product will be compatible but destroying the recognition of the restriction site 
-	Digest BB construct with restriction enzymes SnaBI (blunt end) and NheI
-	Ligate the promoter to BB construct
-	Final product: EcorI-CAG-BBF5-XhoI (XhoI is already in the BB construct)
451CNP construct modification to remove loxp site
-	451CNP is the drug selection marker that is used for knock-in cell outgrowth screening
-	Starting plasmid: 451CNP (flanked by frt sites, but also has one loxp site)
-	Designed 451CNP-frt2 oligo (to exclude lopx site, introduce XhoI):
forward: 5' cgaagttcctattctctagaaagtataggaacttcctcgagg 3'
reverse: 5' gatccctcgaggaagttcctatactttctagagaataggaactt 3'
forward strand: 5' cgaa gttcctattctctagaaagtataggaacttc CTCGAG g 3'
forward strand: 5' (BstBI overhang)-Frt-XhoI-(BamHI overhang) 3'
reverse strand: 3'   tt caaggataagagatctttcatatccttgaag GAGCTC cctag 5'
reverse strand: 5' (BamHI overhang)xxxx XhoI (BstBI overhang) 3'
-	Anneal the forward and reserve oligos from above 
-	Digest 451CNP and the annealed oligos with BstBI and BamHI to remove loxp site and introduce XhoI
-	Ligate the oligo to the 451CNP construct
-	Final product: EcorI-451CNP-BstBI-XhoI-BamHI (EcorI is already in the 451CNP-frt construct)
pBS-up-md-dn modification
-	It is a pBlueScript vector backbone constructed and given by Zhili Rong
-	The construct of the up-mid-down arm is part of the targeting arm of BAC plasmid to do recombination, 
       although the desired recombination event should take place between up and down arm, 
       but there are rare chances to observe recombination happen between up-mid or mid-down arms
-	Insert 451CNP between up and mid arm, then insert CAG-BBF5 between mid and down arm
-	Need to modify md-dn arm segment by introducing few more restriction sites in between, 
       then there will be compatible restriction sites to introduce other BB construct, 
       also having extra restriction sites gives the plasmid more flexiblility for other application/construct design
-	Starting plasmid: up-mid-BamHI-EcorV-down
-	Designed HPRT-md-dn oligo to introduce MfeI, NheI, AgeI between mid and down arm:
forward: 5' gatcccaattggctagcaccggtgtcgacgat
reverse: 5' atcgtcgacaccggtgctagccaattgg
forward strand: 5' gatcc caattg gctagc accggt gtcgac gat
forward strand: 5'(BamHI overhang)MfeI NheI AgeI SalI(EcorV overhang) 3'
reverse strand: 3'     g gttaac cgatcg tggcca cagctg cta
reverse strand: 5'(EcorV overhang)SalI AgeI NheI MfeI(BamHI overhang) 3'
-	Anneal the forward and reverse oligos
-	Digest the pBS-up-mid-down plasmid and the annealed oligos with MfeI and SalI
-	Final product: pBS-up-mid-BamHI- MfeI-NheI-AgeI-SalI-EcorV-down
pBS-up-451CNPfrt-md-dn construction
-	Starting plasmid: pBS-up-MfeI-SalI-mid-down, EcorI-45CNPfrt-BstBI-XhoI-BamHI
-	Restriction enzyme pair (MfeI and EcorI) and (XhoI and SalI) will create same overhang after enzyme digestion, 
       so that they will be compatible for ligation, but the restriction sites will be disrupted after ligation. 
-	It is a good strategy to get rid of certain common restriction sites for complicated construct design to keep these sites for future use/different sites.
-	Digest pBS-up-MfeI-SalI-mid-down with MfeI and SalI
-	Digest EcorI-45CNPfrt-BstBI-XhoI-BamHI with EcoI and XhoI
-	Ligate the two products
-	Final plasmid: pBS-up-451CNPfrt-mid-BamHI-MfeI-NheI-AgeI-SalI-EcorV-down
pBS-up-451CNPfrt-md-BBF5-dn construction
-	Starting plasmid: pBS-up-451CNPfrt-mid-BamHI-MfeI-NheI-AgeI-SalI-EcorV-down, CAG-BBF5 (EcorI-CAG-BBF5-XhoI)
-	Digest CAG-BBF5 with EcorI and XhoI
-	Digest pBS-up-451CNRfrt-mid-down with MfeI and SalI
-	Restriction enzyme pair (MfeI and EcorI) and (XhoI and SalI) will create same overhang after enzyme digestion, 
       so that they will be compatible for ligation, but the restriction sites will be disrupted after ligation. 
-	Ligate the two products
-	Final plasmid: pBS-up-451CNPfrt-mid-CAGBB-down
HPRT-451CNPfrt-BBF5 BAC construction
-	Prepare a fresh batch of BAC competent cells or take out previously made frozen ones
-	Electroporate pBS-up-451CNPfrt-mid-CAGBB-down into SW102 BAC competent cells
-	Recover in 30C for 30-60 min with 900 ml low or no salt LB
-	Plate the supernatant/centrifuged pellet onto agar plates (with Kan/Amp/Chl resistance)
-	Let the colonies grow ~24 hrs or more (the number should be fairly small around 10 or so, not expecting huge spread of colonies), 
       pick the colonies to shake in 3 ml culture o/n
-	Save small aliquot (15% glycerol as final concentration) for backup, extract DNA from the rest of the liquid culture to validate final product
Validation of final BAC product
SpeI
BamHI

BAC mediated Brainbow knock-in PGP1iPS cell construction (12/20/2013 – 4/9/2014)[edit]

Recover PGP1iPS cells
BAC mediated recombineering
Outgrowth screening of BB knock-in cells
flp-mediated excision of puro selection marker
Outgrowth screening of final Brainbow positive cells

feeder-free adaptation and CreER knock-in (4/10/2013 – 8/2014)[edit]

teratoma assay (8/2014-10/2014)[edit]

8-25-2014: inject ~4-5 million PGP1iPS-BB-Cre-2-3-10 cells into NSG mice per site (two sites total on each of the hind leg); 
                  mixed with 50% matrigel to protect the injected cells
8-26-2014: inject ~3-4 million PGP1iPS-WT (one site on the hing leg) and PGP1iPS-BB-Cre-2-5-2 cells into NSG mice per site (two sites total on each of the hind leg); 
                  mixed with 50% matrigel to protect the injected cells
Harvest the Teratoma:
10-5-2014: harvest PGP1iPS-BB-Cre-2-3-10 teratomas
10-17-2014: harvest PGP1iPS-WT and PGP1iPS-BB-Cre-2-5-2 teratomas

NPC and Cardiomyoctye differentiation (10/2014-12/2014)[edit]

NPC differentiation protocol:
Day 0: when the cells are ready to passage (~80% confluent), change to N2 media (1x N2 in DMEM/F12 without antibiotics). 
This is important to get the EB in N2 media ahead of time. Because there will be a lot of cell death and it is too stressful to the cells, if you change on the day that you do EB.

Day 1: change media again for N2 media supplemented with 1uM of Dorsomorphin (Calbiochem, cat #171261) + SB 431532 
NOTE: I have tried to use this as day 0, and the rest as follows; the protocol seems to work fine

Day 2: create the EB – wash the plate with PBS-Ca-Mg; cut the cells into small compartments using yellow tips, add PBS and incubate at 37 C for 5 minutes; 
aspirate the PBS then add in 2 ml of N2 media (+DO +SB), scrap the cells with cell lifter (pay attention to the cells/clumps that are trapped in the cell lifter); 
transfer to low binding plates (4 ml of media for 1 well of 6-well plate).

Day 3 and onward: check the cells next day for possible media change. 
Change the media from EBs at least two days after scrapping the ells and keep changing when the media is yellow or have too many dead cells.

Day 10: plate on matrigel plates using NG+FGF (0.5x N2, 0.5x B27, 20ng/ml FGF and 1% P/S – try to add FGF fresh). Use 1 ml pipette to dissociate the EBs a little bit, 
the rosette will arise from the plated EBs. Dissociate and passage the cells/only rosette using accutase and plate on poliornithine/Laminin plates using NG+FGF media.

Further neuronal differentiation: from 90-100% confluent plate, withdraw FGF form NG media and add Rock inhibitor for 48 hours. 
Change the media with NG only after 48 hours. Keep for at least 4 weeks.
Cardiomyocyte differentiation protocol using Activin A and BMP4:
90% confluent hiPSCs maintained on feeder plate in PGP1iPS media were dissociated into cell clusters with collagenase IV at 37 °C for 5 min,
and then were seeded onto a Matrigel-coated cell-culture dish at 1:3 dilution in media (50% PGP1iPS + 50% PSGro) supplemented with ROCK inhibitor 
(RI, Y-27632; Stemgent) (day -5 ~ -7) for 24 h. 

Cells then were cultured in media (50% PGP1iPS + 50% PSGro) until reach 80-90% confluency, which was changed daily 
Day 0: cells were treated with 4ml of ¾ basal media (1640 RPMI+B27-insulin) + ¼ PSGro + CHIR99021 + RI 1:5000
Day 1: cells were treated with Basal media + RI
Day 3: cells were treated with ¼ old media (collected from day 1, try to avoid dead cells) + ¾ Basal media + IWP4 (800x) + RI
Day 5: cells were treated with Basal media + RI
Day 7-12: cells were treated with 1640 RPMI + B27, and change media every other day (monitor the amount of total cell number and dead cell, 
                change media more frequently if necessary)

CRISPR mediated Brainbow knock-in at the AAVS1 locus[edit]

plasmids to be constructed:
CAG-Cas9-T2AAVS1   (cas9 enzyme and gRNA)
Donor CAG-BB-Puro (donor template for knock-in)

CAG-Cas9-T2AAV1 construction (late 12/2014)[edit]

- Zhili has constructed CAG-Cas9 plasmid with BaeI site that can be digested to add desired gRNA oligo
- use BaeI to cut the plasmid, anneal oligos T2F (sequence: GGGGCCACTAGGGACAGGAT) and T2R (Church lab Addgene plasmid #41818)
- transformation and Sanger (also enzyme digest) validation until obtain the correct final produt

- also got another gRNA plasmid from Jingjin (designed using software, he got ~30% homozygous knock-in)
- CAG-Cas9-GCAMP3 gRNA (the gDNA location is only few basepair away from T2, they are almost identical)
- will try this gRNA to test if there is difference in efficiency

Donor CAG-BB-Puro plasmid construction (1/2015-early 2/2015)[edit]

- Dr. Parshant Mali gifted the plasmid Puro-CAGGSp-EGFP-pA (DNA)
- perform transformation to save glycerol stock and extract gDNA for mini-prep
- validate sequences on some parts of the plasmid (enzyme digest and sanger sequencing)
- use NcoI + MluI to digest the validated clone (NcoI is part of CAG/CMV promoter, and MluI is right after EGFP)
- take existing plasmid CAG-BB-F5
- validate the plasmid before use, use CAGBB insert validate primer: CGT TGG AGT CCA CGT TCT TT
- then design oligos to modify the plasmid to insert into Dr. Mali's donor plasmid

     MluI EcoRI
 5’ acgcgtgaattcactcctca   3’
     tgcgcacttaagtgaggagt
 5’ cgcgtgaattcactcctca ggtgc    3’                     
           Acttaagtgaggagt ccacg
 3’  cgtgg actcctcacttaagtgcgc 5’
 5’  gcacc tgaggagtgaattca        3’

     HindIII           XhoI
5’  aagcttgagctcgag 3’
     ttcgaactcgagctc
3’  gagct cgtggactcctcacttaagttcga a 5’
5’  c tcgagcacctgaggagtgaattca agctt 3’ 

BB-HindIII-oligo: agcttgaattcactcctcaggtgc
BB-XhoI-oligo: tcgagcacctgaggagtgaattca

- use HindIII and XhoI to cut the plasmid
- anneal the above F+R oligos
- ligate the insert into CAG-BB-F5 plasmid --> rename the plasmid CAG-BB-CRISPR
- digest the plasmid CAG-BB-CRISPR with SnaBI and XhoI
- Gibson assembly of the two plasmids:  
1) CAG-BB-CRISPR with SnaBI and XhoI
2) Puro-CAGGSp-EGFP-pA with NcoI and MluI
-final product is then validated with 
donor-HAL-validate F:   5' GCA ACC TCC CCT TCT ACG AG 3'
donorBB-HAR-validate: 5' CCC ATA TGT CCT TCC GAG TG 3'

CRISPR mediated Brinbow knock-in (2/2015-4/2015)[edit]

- Maxi-prep to get large quantity of the plasmids to electroporate into the cells
- pick single colony/little glycerol stock to do 3ml culture overnight at 37C (start early morning before 10 am)
- shake 300 ml of LB overnight (with antibiotics)
- Maxi-prep to extract plasmid DNA
- linearize the donor plasmid with enzyme NsiI overnight 
- wash digested plasmid with the following protocol:
   - prepare 1ml of 100% ethanol, 3 tubes of 1ml 70% ethanol
   - add 2x vol of 100% ethanol, invert mix, then big ball of DNA/pellet should be seen in solution (or precipitated) 
   - use 1ml tip to carefully transfer the pellet into 70% ethanol tube and then to the next tube, repeat for total of 3 washes
   - centrifuge at 15,000 rpm for 5 min
   - dry in culture hood then re-suspend in autoclaved clean H2O
Electroporation:
- since CRISPR construct is much smaller than BAC, only small amount of cells are needed (e.g. 1 well of 6-well plate)
- PBS wash the cells once, then add TrypLE to digest the cells for 5 min at 37C
- aspirate the enzyme, then wash with PBS once (cells should still attach well)
- use 1000ul pipette to dissociate the cells into patches/clusters, transfer to 15/50 tube
- spin down at 1000 rpm for 5 min
- re-suspend with 500ul PBS, swirl mix (DO NOT invert due to low volume) then pipette 5 times
- add appropriate amount of cas9, gRNA, and donor plasmid into the cells
   - example: Cas9 (10kb) - 5ug; gRNA (4kb) - 2ug; donor plasmid (16kb) - 8ug 
   - gRNA+Cas9 AAVST2 or GCAMP3 (9.7kb) - 5ug; donor plasmid (14kb) - 7ug
- bring the cuvette to perform electroporation
   - electroporation setting: 320V, 200uF, (turn knob to max to go above 25uF)
- leave in hood RT to allow the cells to recover for 10 min
- transfer the cells onto matrigel coated plate drop-wise to allow them spread evenly
- the knock-in was done on both PGP1iPS and H9 human iPSC and ESC lines respectively

Flipped-Brainbow knock-in cell line (3/2015-6/2015)[edit]

- we have previously sorted PGP1iPS-BB-2-3-10 line (added Cre-ER) into 4 population (4 colors respectively)
- continous culture and differentiation into 3 germ layer suggested possible toxicity/selection pressure for hrGFP
- we decided to flip the entire cassette to switch the default color to CFP
- using two single digest enzyme that flank GFP+RFP and YFP+CFP, we could flip the two group of XFPs 
- after cloning experiment, then repeat the electroporation and screen for positive clones

cardiomyocyte differentiation and de-differentiation (7/2015-present)[edit]

- general protocol that worked well for PGP1iPS and H1 line differentiation:
   - day 1: CHIR (final is 6uM)
   - day 2: CHIR (final is 6uM)
   - day 3: IWR1 (final is 10uM)
   - day 4: IWR1 (final is 10uM)
   - day 5: 1640 + B27-insulin (basal media)
   - day 6: (basal media)
   - day 7: (basal media)
   - day 8: 1640 + B27 with insulin (so far looks like can culture up to 5 weeks or 40 days)