Abstract

Transcription regulation is a key aspect of cellular identity established during development and maintained into adulthood. Molecular and biochemical assays that probe the genome are critical tools in exploring mechanisms of transcription regulation and cell type identity. The mammalian brain is composed of a huge diversity of cell types with distinct properties and functions. To understand these specific roles, it is necessary to selectively target cell populations for study. However, the need to selectively study restricted cell populations poses a challenge in neurobiology. It is often difficult to collect sufficient cellular input for many standard biochemical and molecular assays. Recently, important advances have been made to scale assays down, opening up new frontiers to explore molecular mechanisms in neurons. Concurrently, methodologies for preparing neurons for such assays has advanced taking into consideration specific methods to preserve the cell biology meant to be assayed. Here we describe a method for preparing live neurons from adult brain tissue for the Assay for Transposase Accessible Chromatin (ATAC).

Highlights

  • [Background] The Assay for Transposase Accessible Chromatin coupled with generation sequencing (ATAC-seq) is becoming a widely used method to assess genome-wide chromatin accessibility (Buenrostro et al, 2013; Corces et al, 2017)

  • While single cell methods play an important role in identifying heterogeneity and defining certain characteristics of cell types, they are often not suitable for deeper, reproducible characterization, or targeted manipulation and comparison of cell types

  • We adapt an earlier protocol for isolating neurons from live, adult brain tissue (Hempel et al, 2007) to generate ATAC-seq profiles from sorted populations of 20,000-50,000 cells expressing a fluorescent marker

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Summary

Procedure

Note: If working with precious small amounts of tissues, rinse the cell strainer cap with the 1 ml and transfer any chunks left in the top back to the Eppendorf for the trituration step. Note: The first cerebellar granule cell pellet typically contains too much debris To clean it up further the pellet is resuspended in another 5 ml of 20% Percoll, and centrifuged again. You may add a Percoll cushion to the bottom of the tube containing the supernatant and centrifuge again This typically results in a pellet with additional cells, but often contains more debris than the first pellet. An additional option is to layer the cell suspension on top of the Percoll solution This has the disadvantage of making it difficult to do additional spins on the supernatant but may allow better fractionation of some samples. The pellet is often not visible for samples < 40,000 nuclei

36. Prepare 1x tagmentation solution
Findings
Lysis buffer
Full Text
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