Jul 19, 2022

Public workspaceSTICR Barcode Library Amplification Protocol

Forked from a private protocol
DOI
dx.doi.org/10.17504/protocols.io.8epv598r4g1b/v1
  • Ryan N. Delgado1,
  • Denise E. Allen1,
  • Matthew G. Keefe1
  • 1University of California at San Francisco, San Francisco, CA, USA
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DOI: dx.doi.org/10.17504/protocols.io.8epv598r4g1b/v1
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
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
PCR
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 Amount30 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 Amount50 µL reaction:
  1. Amount25 µL Q5 High-fidelity 2X Master Mix
  2. Amount2.5 µL Concentration10 micromolar (µM) i7_indexed Reverse Primer (283-290)
  3. Amount2.5 µL Concentration10 micromolar (µM) i5_indexed Forward Primer (291-298)
  4. Amount10 µL molecular grade H20
  5. Amount10 µL 10X cDNA







PCR program:
  1. Temperature98 °C for Duration00:00:30 seconds
  2. Temperature98 °C for Duration00:00:10 seconds
  3. Temperature62 °C forDuration00:00:20 seconds
  4. Temperature72 °C for Duration00:00:10 seconds
  5. 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)
  6. Temperature72 °C for Duration00:02:00 minutes
  7. Temperature4 °C Hold











Post PCR cleanup
Post PCR cleanup
Perform dual-sided SPRI selection
Add Amount30 µL of SPRI beads to Amount50 µL of PCR reaction, mix by pipetting 15 times.


Incubate at RT for Duration00:05:00 minutes

Place on 10X magnet on High for Duration00:03:00 minutes. DO NOT discard supernatant.

Critical
Transfer supernatant to new PCR tube.
Add Amount10 µL of SPRI beads, mix by pipetting 15 times.

Incubate at RT for Duration00:05:00 minutes

Place on 10X magnet on High for Duration00:03:00 minutes

Carefully remove and discard supernatant (do not disrupt beads).
Wash beads with Amount200 µL of freshly prepared 80% EtOH.

Let stand Duration00:00:30 seconds, remove EtOH.

Wash 1 additional time with 80% EtOH for Duration00:00:30 seconds

Remove EtOH with pipette, briefly centrifuge and return PCR tube to 10X magnet in low position. Remove any residual EtOH with pipette.
Add Amount22 µL of Buffer EB, mix by pipetting 15 times.

Let stand Duration00:05:00 minutes RT.

Place on 10X magnet on low for Duration00:03:00 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.