TITLE: iTracer-perturb Plasmid Prep
AUTHORS: Ashley  Maynard, Hsiu-Chuan  Lin

[DESCRIPTION]
Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR–Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaicTSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.

This protocol describes the creation of the iTracer vectors. 

[STEPS]
SECTION: Order iTracer plasmids
1. Please order iTracer from the European Plasmid Repository (https://www.plasmids.eu/).

pSBbi-iTracer-perturb-R2AG-gG
pSBbi-iTracer-perturb-B2AG-gG
SECTION: Order barcodes
31. To ensure the greatest barcode diversity please order the barcode components below:

barcode template: GGACGAGCTGTACAAGTAAGCTGATCCWNNNNNNNNNWCCAAGCgcccttgagcatctgacttc
barcode forward primer: GGACGAGCTGTACAAGTAAGCTG
barcode reverse primer: gaagtcagatgctcaagggcGCTTG
SECTION: Amplify barcodes
32. Resuspend all primers to concentration of 100 micromolar (µM), you can store these stocks at -20 °C

Dilute stock primers to 10 micromolar (µM)
Dilute barcode template 50 micromolar (µM)
SECTION: Amplify barcodes
33. Amplify barcodes by combing the following in a PCR tube:

1.25 µL  diluted barcode forward primer (from step #3)
1.25 µL  diluted barcode reverse primer (from step #3)
0.5 µL  diluted barcode template (from step #3)
9.5 µL water
SECTION: Amplify barcodes
34. Run PCR program

98 °C for  2 min 

(25 cycles total)
Step 5.2: 98 °C for  10 s 
Step 5.3: 58 °C for  20 s 
Step 5.4: 72 °C for  20 s 

 72 °C for  5 min 
 4 °C  until ready to proceed.
SECTION: Amplify barcodes
35. Run 1% agarose gel with 1 µL of amplified barcode product to check for successful amplification. 
SECTION: NotI Digest of iTracer-perturb+guide plasmid
36. To cut the plasmid for barcode insertion first digest the plasmid with NotI-HF by combining in a PCR tube:

5 µg iTracer-perturb+guide plasmid
5 µL Cutsmart buffer (NEB #B7204)
3 µL NotI-HF
fill to 50 µL with water

SECTION: NotI Digest of iTracer-perturb+guide plasmid
37. Incubate 37 °C   

SECTION: NotI Digest of iTracer-perturb+guide plasmid
38. Run 0.5% agarose gel with digested and undigested plasmid (control) to check for complete cutting.

SECTION: NotI Digest of iTracer-perturb+guide plasmid
39. Perform a get extraction clean up (Macherey-Nagel #REF740609.50) of EcoRI fragmented plasmid and nanodrop.
SECTION: Gibson Assembly of Barcodes into iTracer plasmid
41. Combine in a PCR tube:

1ul of the calculated volume of EcoRI fragmented plasmid
1ul of the calculated volume of barcodes from step #15
10 µL NEBuilder®HiFi DNA Assembly Master Mix (NEB #E2621S)
fill to 20 µL with water

** Make sure to set-up a control following the same reaction conditions above but without barcodes, instead add water. We will use this reaction to estimate self ligation of the plasmid.
SECTION: Gibson Assembly of Barcodes into iTracer plasmid
42. Incubate 15 min at 50 °C. Then place on ice.

SECTION: Collect final plasmids
45. To collect Gibson assembled plasmids use 10 mL of 2yt buffer and a scraper to collect all the colonies.

SECTION: Collect final plasmids
46. Wash the plate with5-7 mL 2yt buffer and collect in the same tube as the collection in step #18.
SECTION: Collect final plasmids
47. Perform x3 Qiagen Midiprep (Qiagen #12143) reactions, elute in 200 µL

SECTION: Collect final plasmids
48. Pool final midiprep reactions (final volume ~600 µL) and nanodrop for final concentration.  You now have barcoded iTracer-perturb plasmids ready for electroporation into your cells!

SECTION: Order and amplify the guide donor plasmid
2. Order plasmid: pMJ114 from addgene (https://www.addgene.org/85995/?gclid=CjwKCAjw0a-SBhBkEiwApljU0o4BqNfCIJ0XWMbzs8XS691A3nujA1zPVsyWJjiVOAHEKqRWnAoSQxoCzXoQAvD_BwE)
SECTION: Order custom guide
8. To add your custom guide first order your guide, replacing [YOURgRNA] sections with the sequences you wish to target.

forward: atgg [YOURgRNA] gtttaagagc
reverse: ttagctcttaaac [YOURgRNA(complement and reverse)] ccataaac
SECTION: Order and amplify the guide donor plasmid
3. Amplify pMJ114 by taking a non-filtered p10 tip and dipping into glycerol stock of pMJ114. Add this tip to 3 mL of 2yt buffer + Ampicillin


SECTION: Order and amplify the guide donor plasmid
4. Incubate  (~16hr) at 37 °C. 

SECTION: Digest the pMJ114 plasmid
6. BstXI followed by BlpI digestion (removes the "filler" guide")
SECTION: Order and amplify the guide donor plasmid
5. Harvest the cells from the liquid culture and use a Qiagen Miniprep (Qiagen #27106X4) column to purify the plasmid and elute in 35 µL. Nanodrop the purified plasmid to obtain the concentration.
SECTION: Digest the pMJ114 plasmid
7. Run a 1% agarose gel and preform a gel extraction clean up to obtain a clean plasmid fragment.
SECTION: Ligate your custom gRNA into pMJ114 plasmid
9. Phosphorylate and anneal the oligo pairs from step 8: 


1 µL  10uM forward primer
1 µL  10uM reverse primer
1 µL  of 10x T4-Ligase Buffer
6.5 µL water
0.5 µL T4 PNK (https://international.neb.com/products/m0201-t4-polynucleotide-kinase#Product%20Information)

SECTION: Ligate your custom gRNA into pMJ114 plasmid
10. Ligate (T4 DNA ligase, NEB #M0202S)



100 ng of digested plasmid from step 7
1 µL of phosphorlated and annealed oligo pairs from step 9
3 µL of 10x ligase buffer
1 µL of T4 DNA ligase
add water to 30 µL


Incubate at Room temperature for at least 1hr 

SECTION: Ligate your custom gRNA into pMJ114 plasmid
12. Select a subset of colonies for sequencing for quality control and for mini prep. Here you will split the colony between your choice of sequencing platform to check for incorporation of your guide and amplifying your colony by taking a non-filtered p10 tip and dipping into colony and adding this tip to 3 mL of 2yt buffer + Ampicillin.
SECTION: Ligate your custom gRNA into pMJ114 plasmid
13. Mini prep (Qiagen #27106X4)  the clone(s) that sequencing confirms has the incorporated guide.
SECTION: Ligate your custom gRNA into pMJ114 plasmid
11. Transform component cells with:

50 µL cells + 10 µL reaction mix
on ice 15 min
Heat shock the cells for 45 sat 42 °C
Place tubeon ice for 1 min
Add 350 µLSOC

Incubate by shaking (~ ) for 45 min at 37 °C

Plate cells on Amp plates and incubate overnight at 37 °C
SECTION: Ligate your custom gRNA into pMJ114 plasmid
14. PCR amplify the guide area using:

forward primer: ggggtcggcaattgaaaccggtcgtgaccgagcttgtctgccc
reverse prmer: gggaaataggccctcgcacatcgtgatagtcagactggaaaaaaagcacc

Amplify barcodes by combing the following in a PCR tube:

1.25 µL  10uM forward primer
1.25 µL  10uM reverse primer
1.25 µL  guide template
12.5 µL  of Phusion Master Mix
9.5 µL water

SECTION: Ligate your custom gRNA into pMJ114 plasmid
16. Run a 1% agarose gel and preform a gel extraction clean up to obtain a clean PCR product (guide product).
SECTION: Ligate your custom gRNA into pMJ114 plasmid
15. Run PCR program

98 °C for  2 min 

(25 cycles total)
Step 5.2: 98 °C for  10 s 
Step 5.3: 58 °C for  20 s 
Step 5.4: 72 °C for  20 s 

 72 °C for  5 min 
 4 °C  until ready to proceed.
SECTION: Amplify iTracer-perturb plasmid
19. Harvest the cells from the liquid culture and use a Qiagen Miniprep (Qiagen #27106X4) column to purify the plasmid and elute in 35 µL. Nanodrop the purified plasmid to obtain the concentration. 

SECTION: Amplify iTracer-perturb plasmid
18. Incubate  (~16hr) at 37 °C. 

SECTION: Amplify iTracer-perturb plasmid
17. Transform component cells with:

50 µL cells + 2 µL plasmid
on ice 15 min
Heat shock the cells for 45 sat 42 °C
Place tubeon ice for 1 min
Add 350 µLSOC
Incubate by shaking (~ ) for 45 min at 37 °C

Add reaction mix to 3 mL of 2yt buffer + Ampicillin
SECTION: AgeI digest of iTracer-perturb plasmid
20. To cut the plasmid for guide-product insertion first digest the plasmid with AgeI by combining in a PCR tube:

5 µg iTracer-perturb plasmid
5 µL Cutsmart buffer (NEB #B7204)
3 µL AgeI
fill to 50 µL with water

SECTION: AgeI digest of iTracer-perturb plasmid
21. Incubate 37 °C   

SECTION: AgeI digest of iTracer-perturb plasmid
22. Run 1% agarose gel with digested and undigested plasmid (control) to check for complete cutting.

SECTION: AgeI digest of iTracer-perturb plasmid
23. Perform a column clean up (Macherey-Nagel #REF740609.50) of AgeI fragmented plasmid and nanodrop.
SECTION: Gibson assembly of guide product and iTracer-perturb
27. 50 µL cells + 10 µL reaction mix
on ice 15 min
Heat shock the cells for 45 sat 42 °C
Place tubeon ice for 1 min
Add 350 µLSOC

Incubate by shaking (~ ) for 45 min at 37 °C
SECTION: Gibson assembly of guide product and iTracer-perturb
28. Plate the reaction volume on Ampicillin plates and incubate   at 37 °C
Plate the control reaction on Ampicillin plates and incubate   at 37 °C
SECTION: Gibson assembly of guide product and iTracer-perturb
24. Calculate the pmol of 1ul of the guide-product and the pmol of 1ul of the iTracer-perturb plasmid fragment (this calculator can help https://ch.promega.com/resources/tools/biomath/)

Example:

guide amplicon = 500bp with 0.18ug/ul = 0.55pmol DNA  (in 1ul)
plasmid fragment = 5613bp with 0.2044ug/ul = 0.055pmol  (in 1ul)

guide product should be 10 fold the molar excess of an insert, therefore:
10 x 0.055 pmol (the plasmid fragment) = 0.55 pmol (the molarity we need of our barcodes)

In this example we will use 1ul of guide-product for 1ul of vector in the gibson assembly
SECTION: Gibson assembly of guide product and iTracer-perturb
29. Select a subset of colonies for sequencing for quality control and for mini prep. Here you will split the colony between your choice of sequencing platform to check for incorporation of your guide and amplifying your colony by taking a non-filtered p10 tip and dipping into colony and adding this tip to 3 mL of 2yt buffer + Ampicillin.
SECTION: Gibson assembly of guide product and iTracer-perturb
30. Mini prep (Qiagen #27106X4)  the clone(s) that sequencing confirms has the incorporated guide. (iTracer-perturb+guide plasmid)
SECTION: Gibson Assembly of Barcodes into iTracer plasmid
40. Calculate the pmol of 1ul of the barcodes (step #7) and the pmol of 1ul of the plasmid fragment (step #13) (this calculator can help https://ch.promega.com/resources/tools/biomath/)

Example:

barcodes = 63bp with 0.0238ug/ul = 0.57pmol DNA  (in 1ul)
plasmid fragment (iTracer-R) = 5613bp with 0.2044ug/ul = 0.055pmol  (in 1ul)

barcodes should be 10 fold the molar excess of an insert, therefore:
10 x 0.055 pmol (the plasmid fragment) = 0.55 pmol (the molarity we need of our barcodes)

In this example we will use 1ul of barcodes for 1ul of vector in the gibson assembly
SECTION: Gibson assembly of guide product and iTracer-perturb
25. Combine in a PCR tube:

calculated amount of guide-product (shown above)
1uL iTracer-perturb (AgeI fragmented plasmid)
10 µLGibson Assembly Mix (NEB #E2611S)
fill to 20 µL with water

** control run same reaction without guide product.
SECTION: Gibson assembly of guide product and iTracer-perturb
26. Incubate 15 min at 50 °C. Then place on ice.
SECTION: Gibson Assembly of Barcodes into iTracer plasmid
43. 50 µL cells + 10 µL reaction mix
on ice 15 min
Heat shock the cells for 45 sat 42 °C
Place tubeon ice for 1 min
Add 350 µLSOC

Incubate by shaking (~ ) for 45 min at 37 °C
SECTION: Gibson Assembly of Barcodes into iTracer plasmid
44. Plate the total reaction volume on Ampicillin plates (x3 15cm square plates) and incubate   at 37 °C
Plate 30% of total volume of the control reaction (to be comparable to experimental above) and incubate   at 37 °C