Jul 19, 2022
STICR Barcode Library Amplification Protocol
Forked from a private protocol
- Ryan N. Delgado1,
- Denise E. Allen1,
- Matthew G. Keefe1
- 1University of California at San Francisco, San Francisco, CA, USA
Protocol Citation: Ryan N. Delgado, Denise E. Allen, Matthew G. Keefe 2022. STICR Barcode Library Amplification Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv598r4g1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: July 19, 2022
Last Modified: July 19, 2022
Protocol Integer ID: 67005
Keywords: sticr barcode library amplification protocol barcode amplification protocol, snicr library, human cortical progenitor, individual human cortical progenitor, amplification, inhibitory neuron, excitatory
Abstract
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
Troubleshooting
PCR
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.
PCR Reaction Mix
Amplify library with standard NEB Protocol for Q5 Hot Start High-Fidelity 2X Master Mix in 50 µL reaction:
- 25 µL Q5 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
PCR program:
- 98 °C for 00:00:30 seconds
- 98 °C for 00:00:10 seconds
- 62 °C for00:00:20 seconds
- 72 °C for 00:00:10 seconds
- 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 00:02:00 minutes
- 4 °C Hold
Post PCR cleanup
Perform dual-sided SPRI selection
Add 30 µL of SPRI beads to 50 µL of PCR reaction, mix by pipetting 15 times.
Incubate at RT for 00:05:00 minutes
Place on 10X magnet on High for 00:03:00 minutes. DO NOT discard supernatant.
Transfer supernatant to new PCR tube.
Add 10 µL of SPRI beads, mix by pipetting 15 times.
Incubate at RT for 00:05:00 minutes
Place on 10X magnet on High for 00:03:00 minutes
Carefully remove and discard supernatant (do not disrupt beads).
Wash beads with 200 µL of freshly prepared 80% EtOH.
Let stand 00:00:30 seconds, remove EtOH.
Wash 1 additional time with 80% EtOH for 00:00:30 seconds
Remove EtOH with pipette, briefly centrifuge and return PCR tube to 10X magnet in low position. Remove any residual EtOH with pipette.
Add 22 µL of Buffer EB, mix by pipetting 15 times.
Let stand 00:05:00 minutes RT.
Place on 10X magnet on low for 00:03:00 minutes
Remove supernatant to new PCR tube. This is your barcode library.
You can confirm library prep with Agilent BioA/Tapestation.Trace should look like:
Ensure that separate libraries were prepared with different antibodies before pooling and submit for sequencing ~30 million reads per library.