TITLE: STICR Barcode Library Amplification Protocol
AUTHORS: Ryan N. Delgado, Denise E. Allen, Matthew G. Keefe

[DESCRIPTION]
Barcode amplification protocol for SNICR libraries based on:
Delgado, R.N., Allen, D.E., Keefe, M.G.et al.Individual human cortical progenitors can produce excitatory and inhibitory neurons.Nature601,397–403 (2022). https://doi.org/10.1038/s41586-021-04230-7

[STEPS]
SECTION: PCR
1. Following cDNA amplification in 10X workflow, bead purify as directed in instructions and set aside 10ul of cDNA to use in the following reaction. This should leave you with 30 µL of bead purified cDNA to complete the rest of the standard 10X whole transcriptome library with. 

Primers: Reverse primer (P5-Read1) plus a forward primer (P7-i7 index-Read2-Upstream_Barcode sequence). If you plan to sequence multiple barcode libraries on the same lane, you will need to use different reverse primers as they will need to have different i7 indexes.

SECTION: PCR
2. PCR Reaction Mix
Amplify library with standard NEB Protocol for Q5 Hot Start High-Fidelity 2X Master Mix in 50 µL reaction: 
 25 µLQ5 High-fidelity 2X Master Mix
 2.5 µL 10 micromolar (µM) i7_indexed Reverse Primer (283-290)  
  2.5 µL 10 micromolar (µM)i5_indexed Forward Primer (291-298)
 10 µL molecular grade H20
 10 µL 10X cDNA 

 
 






SECTION: PCR
3. PCR program:
98 °C for 30 sseconds
98 °C for 10 sseconds
62 °C for20 sseconds
72 °C for 10 sseconds
Repeat steps 2 through 4 ~15X (if unsure, run tests with 2uL starting cDNA at a range of cycles, but do not exceed 20 cycles for final preparation)
72 °C for 2 minminutes
4 °CHold











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4. Perform dual-sided SPRI selection 
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5. Add 30 µL of SPRI beads to 50 µL of PCR reaction, mix by pipetting 15 times.


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6. Incubate at RT for 5 minminutes

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7. Place on 10X magnet on High for 3 minminutes. DO NOT discard supernatant.

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8. Transfer supernatant to new PCR tube.
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9. Add 10 µL of SPRI beads, mix by pipetting 15 times.

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10. Incubate at RT for 5 minminutes

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11. Place on 10X magnet on High for 3 minminutes

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12. Carefully remove and discard supernatant (do not disrupt beads).
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13. Wash beads with 200 µL of freshly prepared 80% EtOH.

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14. Let stand 30 sseconds, remove EtOH.

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15. Wash 1 additional time with 80% EtOH for 30 sseconds

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16. Remove EtOH with pipette, briefly centrifuge and return PCR tube to 10X magnet in low position. Remove any residual EtOH with pipette. 
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17. Add 22 µL of Buffer EB, mix by pipetting 15 times.

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18. Let stand 5 minminutes RT.

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19. Place on 10X magnet on low for 3 minminutes

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20. Remove supernatant to new PCR tube. This is your barcode library.
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21. You can confirm library prep with Agilent BioA/Tapestation.Trace should look like: 
   
  

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22. Ensure that separate libraries were prepared with different antibodies before pooling and submit for sequencing ~30 million reads per library.