Abstract
The Patch-seq approach is a powerful variation of the patch-clamp technique that allows for the combined electrophysiological, morphological, and transcriptomic characterization of individual neurons. To generate Patch-seq datasets at scale, we identified and refined key factors that contribute to the efficient collection of high-quality data. We developed patch-clamp electrophysiology software with analysis functions specifically designed to automate acquisition with online quality control. We recognized the importance of extracting the nucleus for transcriptomic success and maximizing membrane integrity during nucleus extraction for morphology success. The protocol is generalizable to different species and brain regions, as demonstrated by capturing multimodal data from human and macaque brain slices. The protocol, analysis and acquisition software are compiled at https://github.com/AllenInstitute/patchseqtools. This resource can be used by individual labs to generate data across diverse mammalian species and that is compatible with large publicly available Patch-seq datasets.
Data availability
The data used in this manuscript, the software packages, the detailed protocol, and online resources are freely available to the public and have been consolidated at https://github.com/AllenInstitute/patchseqtools.
Article and author information
Author details
Funding
NIH Office of the Director (P51OD010425)
- Brian E Kalmbach
- Jonathan T Ting
National Center for Advancing Translational Sciences (UL1TR000423)
- Brian E Kalmbach
- Jonathan T Ting
National Institute of Mental Health (U01 MH114812-02)
- Ed Lein
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: The animal research in this study was performed in accordance with the Guide for the Care and Use of Laboratory Animals and the Public Health Service Policy on Humane Care and Use of Laboratory Animals in compliance with National Institutes of Health policy. All housing, handling, and experimental use of the animals occurred with the oversight and approval of the Allen Institute Institutional Animal Care and Use Committee (Protocol 1809). All surgeries and retro-orbital injections were performed under isoflurane anesthesia with perioperative analgesics and fluid support.
Human subjects: De-identified human brain tissue and data used in this research was collected by local hospitals during clinically necessary surgery. Study participants gave informed consent to share their de-identified tissue and data either with the Allen Institute specifically or more broadly with collaborators of the study PIs prior to surgery. Participants consented to share their de-identified genomic data in controlled access in compliance with National Institutes of Health Genomic Data Sharing policy. The study participants were informed that the resulting data might be broadly shared, through publications, presentations, or scientific repositories and of the potential risks of sharing these data. Samples obtained from the Swedish Neuroscience Institute were collected under approved Western Institutional Review Board protocols (#1111798 and #1068035) in collaboration with Drs. Charles Cobb and Ryder Gwinn respectively. Samples obtained from Harborview Medical Center were obtained under approval of the University of Washington Institutional Review Board protocol (#HSD No. 49119) in collaboration with Dr. Jeffrey Ojemann.
Reviewing Editor
- Sacha B Nelson, Brandeis University, United States
Publication history
- Received: December 6, 2020
- Preprint posted: May 24, 2021 (view preprint)
- Accepted: August 12, 2021
- Accepted Manuscript published: August 13, 2021 (version 1)
- Version of Record published: September 9, 2021 (version 2)
Copyright
© 2021, Lee et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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