TITLE: Amplify iTracer Barcode and Scars from 10x cDNA
AUTHORS: Ashley  Maynard, Sophie  Jansen, Giovanna  Brancati

[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 barcode and scar detection from single-cell cDNA:

Barcode and scar regions were amplified from 60-70ng of cDNA remaining from the single-cell RNAseq preparation with three separate PCR reactions. First cDNA was amplified via PCR broadly targeting a region containing both the scar and barcode. Subsequently, the reaction was split equally and we performed a nested PCR separately targeting the barcode and scar regions. Lastly, we added Illumina sequencing adapters (10x Genomics). Following every PCR reaction the samples were cleaned-up using magnetic beads (Beckman Coulter). Libraries are then ready to be sequenced on Illumina sequencer.


[STEPS]
SECTION: Set up Reaction 1 (Root PCR)
1. Make reaction master mix: 
      Component      1x rxn      17x rxn        2x   Phusion Ready Mix      25          10uM 10x Root PCR GFP F Primer      2.5          10uM 10x Universal R Primer      2.5          100% DMSO      1.5               EvaGreen       0.75               cDNA product   (50ng)      17.75 total   
 
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2. Add 30.75 µL per well of reaction master mix to qPCR plates, then add a total of 17.75 µL of sample.

SECTION: Set up Reaction 2a (Nested PCR for Barcodes)
7. Make reaction master mix: 
      Component      1x rxn        2x Phusion Ready   Mix      25        10uM Nest   Barcode GFP Primer      2.5        10uM 10x   Universal R Primer      2.5        EvaGreen       0.75        H20       14.25        Root PCR Product      5   
 
SECTION: Set up Reaction 2a (Nested PCR for Barcodes)
8. Add 45 µL per well of reaction master mix to qPCR plates
SECTION: Set up Reaction 2a (Nested PCR for Barcodes)
9. Run PCR according to the following program:
 
     Step      Temperature      Duration      Cycles        Initial   denaturation      98 ºC      30 sec      1        Denaturation      98 ºC      15 sec      45 cycles & stop when saturated           Annealing      66 ºC      15 sec        Extension      72 ºC      20 sec        Final extension      72 ºC      2 min      1   
Volume 50 µL + 105 °C lid temperature
 
SECTION: Set up Reaction 2a (Nested PCR for Barcodes)
10. Stop reaction in the exponential phase before the curve levels off.
SECTION: Set up Reaction 2a (Nested PCR for Barcodes)
11. Clean up reaction with 1:1 SPRI beads:

SECTION: Set up Reaction 1 (Root PCR)
3. Run PCR according to the following program:
 
 
 
     Step      Temperature      Duration      Cycles        Initial   denaturation      98 ºC      30 sec      1        Denaturation      98 ºC      15 sec      45 cycles & stop when saturated           Annealing      66 ºC      15 sec        Extension      72 ºC      20 sec        Final extension      72 ºC      2 min      1   
Volume 50 µL + 105 °Clid temperature.
SECTION: Set up Reaction 1 (Root PCR)
4. Stop reaction in the exponential phase before the curve levels off (see example)

  

SECTION: Set up Reaction 1 (Root PCR)
5. Clean up reaction with 1:1 SPRI beads (50 µL beads added). Elute in 30 µL EB buffer

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6. Check concentrations of reactions on Nanodrop.
SECTION: Set up Reaction 2a (Nested PCR for Barcodes)
12. Check concentrations of reactions on Nanodrop.
SECTION: Set up Reaction 2b (Nested PCR for Scars)
13. Make reaction master mix: 
 
 
      Component      1x rxn        2x Phusion Ready   Mix      25        10uM Nest   Barcode GFP Primer      2.5        10uM 10x   Universal R Primer      2.5        EvaGreen       0.75        H20       14.25        Root PCR Product      5   
 
 
 
SECTION: Set up Reaction 2b (Nested PCR for Scars)
14. Add 45 µL per well of reaction master mix to qPCR plates.
SECTION: Set up Reaction 2b (Nested PCR for Scars)
15. Run PCR according to the following program:
 
 
 
     Step      Temperature      Duration      Cycles        Initial   denaturation      98 ºC      30 sec      1        Denaturation      98 ºC      15 sec      45 cycles & stop when saturated           Annealing      66 ºC      15 sec        Extension      72 ºC      20 sec        Final extension      72 ºC      2 min      1   
Volume 50 µL + 105 °Clid temperature
 
 
SECTION: Set up Reaction 2b (Nested PCR for Scars)
16. Stop reaction in the exponential phase before the curve levels off.
SECTION: Set up Reaction 2b (Nested PCR for Scars)
17. Clean up reaction with 1:1 SPRI beads:
SECTION: Set up Reaction 2b (Nested PCR for Scars)
18. Check concentrations of reactions on Nanodrop.
SECTION: Reaction 3 (Indexing PCR with 10x indexes)
19. Prepare reaction mix:
 
     Component      1x rxn      34x rxn        2x Phusion Ready   Mix      25ul          10x SI Primer       1ul          10x Index   primers      5ul        60ng of PCR   product      19ul total   
 
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20. Prepare the DNA+H20, normalize PCR products to 60 ng, and fill to 19 µL with water 

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21. Add 26 µL of reaction mix to each sample.

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22. Add 10x Index primers 5 µL.

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23. Run PCR according to the following program:
     Step      Temperature      Duration      Cycles        Initial   denaturation      98 ºC      30 sec      1        Denaturation      98 ºC      15 sec      45 cycles & stop when saturated           Annealing      66 ºC      15 sec        Extension      72 ºC      20 sec        Final extension      72 ºC      2 min      1   
Volume 50 µL + 105 °C lid temperature
 
 
 
 
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24. SPRI Select bead clean-up (double sided clean-up as performed at 10x final clean up, v3.1 revD step 3.6) 
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24.1. Add 50 µL of EB to the PCR reactions to have 100 µL total volume.

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None. EN-GB'>Place the magnet•High until the solution clears. DO NOT discard supernatant.
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None. EN-GB'>Transfer 150 μl supernatant to a new tube strip. 
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None. EN-GB'>Vortex to resuspend the SPRIselect reagent. Add 20 μl SPRIselect Reagent
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None. (0.8X) to each sample. Pipette mix 15x (pipette set to 150 μl). 
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None. EN-GB'>Incubate 5 min at room temperature. 
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None. 8. 
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None. EN-GB'>Place the magnet•High until the solution clears. 
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None. 9. 
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None. EN-GB'>Remove 165 μl supernatant. DO NOT discard any beads. 
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None. 10. 
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None. EN-GB'>With the tube still in the magnet, add 200 μl 80% ethanol to the pellet.
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None. Wait 30 sec. 
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None. 11. 
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None. EN-GB'>Remove the ethanol. 
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None. EN-GB'>Repeat steps i and j for a total of 2 washes. 
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None. EN-GB'>Centrifuge briefly. Place on the magnet•Low. Remove remaining ethanol. 
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None. 14. 
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None. EN-GB'>Remove from the magnet. Add 35.5 μl Buffer EB. Pipette mix 15x. 
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None. 15. 
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None. EN-GB'>Incubate 2 min at room temperature. 
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None. EN-GB'>Place on the magnet•Low until the solution clears. 
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None. 17. 
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None. EN-GB'>Transfer 35 μl to a new tube strip. 
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None. EN-GB'>Store at 4°C for up to 72 h or at −20°C for long-term storage 
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None. EN-GB'>Run Bioanalyzer (1:10)
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24.2. Vortex to resuspend the SPRIselect reagent. Add 60 µL SPRIselect Reagent (0.6X) to each sample. Pipette mix 15x (pipette set to 150 µL). Incubate 5 min at 105 Room temperature. 






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24.3. Place the magnet on High until the solution clears. DO NOT discard supernatant. Transfer 150 µL supernatant to a new tube strip. 

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24.4. Vortex to resuspend the SPRIselect reagent. Add 20 µL SPRIselect Reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 150 µL). Incubate 5 min at 105 Room temperature. 

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24.5. Place the magnet on High until the solution clears. Remove 165 µLsupernatant. DO NOT discard any beads. 

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24.6. With the tube still in the magnet, add 200 µL 80% ethanol to the pellet. Wait30 s. Remove the ethanol. Repeat steps i and j for a total of 2 washes. 

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24.7. Centrifuge briefly. Place on the magnet on Low. Remove remaining ethanol. 
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24.8. Remove from the magnet. Add 35.5 µL Buffer EB. Pipette mix 15x. Incubate 2 min at 105 Room temperature. 

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24.9. Place on the magnet on Low until the solution clears. Transfer 35 µLto a new tube strip. 

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24.10. Run Bioanalyzer (1:10 diluted).

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24.11. Store at 4 °C for up to 4320 min or at -20 °C for long-term storage. Libraries are ready for sequencing!

SECTION: Set up Reaction 2b (Nested PCR for Scars)
17.1. Vortex to resuspend the SPRIselect reagent. Add 50 µL SPRIselect Reagent (1X) to each sample. Pipette mix 15x (pipette set to 150 µL). Incubate 5 min at 105 Room temperature. 

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17.2. Place the magnet on High until the solution clears. Remove 165 µLsupernatant. DO NOT discard any beads. 

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17.3. With the tube still in the magnet, add 200 µL 80% ethanol to the pellet. Wait30 s. Remove the ethanol. Repeat steps i and j for a total of 2 washes. 

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17.4. Centrifuge briefly. Place on the magnet on Low. Remove remaining ethanol. 
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17.5. Remove from the magnet. Add 30 µL Buffer EB. Pipette mix 15x. Incubate 2 min at 105 Room temperature. 

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17.6. Place on the magnet on Low until the solution clears. Transfer 30 µLto a new tube strip. 

SECTION: Set up Reaction 2a (Nested PCR for Barcodes)
11.1. Vortex to resuspend the SPRIselect reagent. Add 50 µL SPRIselect Reagent (1X) to each sample. Pipette mix 15x (pipette set to 150 µL). Incubate 5 min at 105 Room temperature. 

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11.2. Place the magnet on High until the solution clears. Remove 165 µLsupernatant. DO NOT discard any beads. 

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11.3. With the tube still in the magnet, add 200 µL 80% ethanol to the pellet. Wait30 s. Remove the ethanol. Repeat steps i and j for a total of 2 washes. 

SECTION: Set up Reaction 2a (Nested PCR for Barcodes)
11.4. Centrifuge briefly. Place on the magnet on Low. Remove remaining ethanol. 
SECTION: Set up Reaction 2a (Nested PCR for Barcodes)
11.5. Remove from the magnet. Add 30 µL Buffer EB. Pipette mix 15x. Incubate 2 min at 105 Room temperature. 

SECTION: Set up Reaction 2a (Nested PCR for Barcodes)
11.6. Place on the magnet on Low until the solution clears. Transfer 30 µLto a new tube strip.