Several colleagues have asked for our protocol for preparing low-input samples for single nucleus multiome (gene expression + ATAC) sequencing. It is now available on @STARProtocols ( https://t.co/ozwW4SOWkP) so that everyone can give it a try. 1/7
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Single-cell/nucleus sequencing has been increasingly used to study specific cell groups such as molecularly defined neurons. Yet, during isolation steps like FACS, the small number of cells/nuclei often gets diluted and is difficult to reconcentrate via centrifugation. 2/7
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In a 10X Genomics multiome assay, where only 5 µL of samples can be loaded, failure to concentrate cell/nuclei not only compromises data quality but necessitates additional sample pooling, resulting in extra time and cost. 3/7
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To overcome this, we have developed a method for cell/nucleus retrieval and enrichment using magnetic beads conjugated with concanavalin A (ConA), which binds to glycoproteins on the surfaces of cells/nuclei. This method offers several advantages over centrifugation. 4/7
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First, it enables the recovery of nearly all nuclei after FACS, which can then be resuspended in a small volume. Second, isolation with magnetic beads is much gentler with minimal loss or damage to nuclei. Third, the simple step adds only 30 min to the existing workflow. 5/7
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Notably, our protocol incorporates additional steps for sample enrichment, multiplexing, and data analyses. Sequencing results revealed an average of 3,644 genes and 13,547 ATAC peaks per nucleus, surpassing those generated using standard protocols. 6/7
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Kudos to the most talented @christ_lili @UTSWInternalMed for developing this with @Baijie_Xu. Special thanks to the Pool lab @Allan_HPool @UTSWNeurosci @UTSWBrain for being among the first to test it in other cell types. We welcome feedback to further improve this protocol! 7/7
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@ChenLiuLab @STARProtocols This looks amazing! Is the data from this study available to download? Would love to compare with some of our data
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@dylanrausch @STARProtocols Thank you, Dylan. We've chosen to publish the protocol/QC data first to share it with the community sooner. The full data set will be part of @Chrsit_lili's upcoming story. For specific inquiries, please reach out to him directly.
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@ChenLiuLab @STARProtocols Great work specially with improving the number of ATAC peaks...Do you think this improved data quality is because of higher nuclei quality? Were you able to visually see the better nuclei quality under the microscope compared to centrifuge way?
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@SepidehSheybani @STARProtocols Thank you, Sepideh! Feel free to visit our office and enjoy some chocolate candies. @christ_lili will be happy to answer any questions you might have.
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@ChenLiuLab @STARProtocols Thanks for this. Our first multiome was not great probably because of low recovery of nuclei.
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@ChenLiuLab @STARProtocols Thanks for sharing the protocol! So totalseq-A hashtag is compatible with multiome kit?
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