TITLE: iTracer 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-G
pSBbi-iTracer-R
SECTION: Order barcodes
2. To ensure the greatest barcode diversity please order the barcode components below:

barcode template: gacgagctgtacaagtgatccgWNNNNNNNNNWcacccagctttcttgtacaaagtggctgtg
barcode forward primer: gacgagctgtacaagtgatc
barcode reverse primer: cacagccactttgtacaaga
SECTION: Amplify barcodes
3. Resuspend all oligos 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
4. 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
5. Run PCR program

Step 5.1: 98 °C for  2 min 

Step 5.2: 98 °C for  10 s 
Step 5.3: 58 °C for  20 s 
Step 5.4: 72 °C for  20 s 
Step 5.5: Repeat steps #5.2-#5.4 x24 times (25 cycles total)

Step 5.6: 72 °C for  5 min 
Step 5.7: 4 °C  until ready to proceed.
SECTION: Amplify barcodes
6. Run 1% agarose gel with 1 µL of amplified barcode product to check for successful amplification. 

SECTION: Amplify plasmids
8. 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: Amplify plasmids
9. Incubate  (~16hr) at 37 °C. 

SECTION: Amplify plasmids
10. 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: EcoRI Digest of iTracer plasmid
11. To cut the plasmid for barcode insertion first digest the plasmid with EcoRI-HF (NEB #R3101S) by combining in a PCR tube:

5 µg iTracer plasmid (from step #9)
5 µL Cutsmart buffer (NEB #B7204)
3 µL EcoRI-HF (NEB #R3101S)
fill to 50 µL with water

SECTION: EcoRI Digest of iTracer plasmid
12. Incubate 37 °C   

SECTION: EcoRI Digest of iTracer plasmid
13. Run 0.5% agarose gel with digested and undigested plasmid (control) to check for complete cutting.

  

SECTION: EcoRI Digest of iTracer plasmid
14. Perform a gel extraction clean up (Macherey-Nagel #REF740609.50) of EcoRI fragmented plasmid and nanodrop.
SECTION: Gibson Assembly of Barcodes into iTracer plasmid
15. 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 Barcodes into iTracer plasmid
17. Incubate 15 min at 50 °C. Then place on ice.

SECTION: Prepare transformation reactions
18. For both the control and plasmid + barcode reactions (preform x2 reactions for plasmid + barcode and x1 reaction for control):

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 for 45 min at 37 °C
SECTION: Prepare transformation reactions
19. For the plasmid + barcode reaction: combine the reaction and equally plate the total reaction volume (~273uL each) on Ampicillin plates (x3 15cm square plates). Add extra SOC buffer to help evenly spread the volume on the plate.

For the control plasmid only reaction: plate ~273ul of total reaction volume, add extra SOC buffer to help evenly spread the volume on the plate.

 Incubate plates 960 min (~16hrat 37 °C

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

SECTION: Collect final plasmids
22. Wash the plate with5-7 mL 2yt buffer and collect in the same tube as the collection in step #18.
SECTION: Collect final plasmids
23. Perform x2 Qiagen Midiprep (Qiagen #12143) reactions, elute in 200 µL

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

SECTION: Collect final plasmids
20. Inspect control plate vs plasmid+barcode plates. There should be a notable difference, where the control plate has few colonies compared to the plasmid+barcode reaction plates. If this is observed continue to step #19.

If this is not the case you may have an error and will need to troubleshoot (control plates with many colonies may have plasmid carryover from from the backbone fragment clean up Step #14, do these steps and repeat). 
SECTION: Gibson Assembly of Barcodes into iTracer plasmid
16. 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: Amplify barcodes
7. Clean up barcodes with column clean up (Macherey-Nagel #REF740609.50) and nanodrop.