ATAC-seq on Sorted Adult Mouse Neurons

[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).

Note: This is a protectant for tissue dissociation (Saxena et al., 2012). The effects of Trehalose are a bit controversial (Lee et al., 2018), but we found it increases the number of intact neurons after trituration.
3. Stir at room temp (RT) and oxygenate using gas diffuser until Kynurenic acid dissolves.
Note: mACSF can remain at room temp.
a. Move enough CS to a new container to oxygenate and suspend one or more cell strainers. i.
Place container in a water bath at 34 °C.
ii. After sectioning, brain slices will recover at 34 °C (Ting et al., 2014 e. Quickly remove brain and place in oxygenating CS filled vibratome buffer tray. Note: For dissection details, see Papouin and Haydon (2018).
f. If slicing multiple brains repeat (c)-(f) for each animal.
g. Make sure brain(s) in tray do not come in direct contact with O2 bubbles.
h. Allow the last brain to equilibrate for 5 min.
a. Place wet blot paper with oxygenated cold CS inside a 100-mm petri dish.
b. Place brain(s) on wetted paper.
c. Remove unwanted tissue and cut to create a flat surface to affix the brain.
i. Examples of two options for mounting the brain are shown in Video 1. The first brain is mounted by removing the cerebellum so that rostral structures will be sliced first. The second brain is oriented in the opposite direction so that caudal structures may be sliced.
Both orientations will produce coronal slices.
ii. Use a fine scalpel to minimize tissue distortion that can damage neurons. 8 www.bio-protocol.org/e3382  Affix the brain in an orientation such that cells of interest are away from the Loctite. e. Move platform back to oxygenated CS.
Note: There is no need to wait for Loctite to air dry.

Micro Dissection and Tissue Digestion
6. Slice mounted brain(s) into 300 µm sections through the region of interest.
a. Set speed to 3 (on a scale of 0-10) and frequency between 6 and 8 (on a scale of 0-10).
Note: Settings should be optimized on individual equipment to minimize tissue distortion/compression. If brains tend to come loose during slicing, then the speed may be too fast or the frequency too low.
b. Reducing tissue deformation/compression will increase yield. We recommend always using a fresh blade. 7. Using a wide mouth pipette or a plastic transfer pipette that has been cut to make the opening larger gently move slices to cell strainers in oxygenated CS at 34 °C a. The strainer protects slices from coming into direct contact with oxygenation bubbles.
b. If working with cells expressing a fluorescent marker, protect samples from light as much as possible from here on to minimize photobleaching. c. Incubate for 10-12 min.  c. The pipette mouth should not be held against the wall or floor of the tube.
d. The solution should turn cloudy with large chunks remaining. 18. Incubate tube(s) on ice allowing chunks to settle for a few minutes. 19. Filter supernatant using tube with cell strainer cap.
Note: You may need to prewet the strainer with mACSF + FBS to start gravity flow.

Hoechst will penetrate live cells. If using other dyes such as DAPI or propidium iodide
Triton x 100 will need to be added to permeabilize cells. 12 www.bio-protocol.org/e3382  ii.
Batching the sample reduces the overall time early sorted cells sit in suspension.
Batches can be pooled later for library prep as needed, prioritizing batches collected earlier in the sort as these tended to give higher quality ATAC.

Pellet sorted cells at 4 °C.
a. Centrifuge sorted cells at 500 x g for 2 min.
b. Rotate tube 180° in holder, centrifuge again at 700 x g for 1 min.
c. Rotate tube 180° again and centrifuge at 500 x g for 2 min.

Note: Alternating the force angle helps collect cells off the tube wall and reduces cell loss
when removing the supernatant.
d. The pellet is often not visible for samples < 20,000 cells.  c. Elute in 6 μl of low TE twice (total volume will be ~11 μl).

Quality Assessment
48. Move 1 μl to a new tube and dilute 1:2 with low TE. Use this aliquot for quality control testing and quantification.
a. Run 1 μl on a High Sensitivity Bioanalyzer Chip to examine the size distribution of fragments Note: Figure 2A shows examples of the size distribution diversity from libraries that gave high quality sequencing data. While nucleosome laddering can indicate a high-quality library, good libraries do not always have this pattern.
b. Use 0.5 μl for qPCR with primers against genomic regions as in Table 2.  b. When considering fragments versus reads it is important to keep in mind that the ends of fragments or one end of a read represents the location of Tn5 access to the genome while the fragment itself is the intervening DNA between two transposition events.

Data Analysis
There are numerous options for analysis of ATAC-seq data that depend on the project goals. As with any project involving bioinformatic analysis it is strongly advised to consult with a statistician or bioinformatician to assess the data needed to achieve project goals. This section is meant to provide www.bio-protocol.org/e3382  basic examples of early analytical steps to aid in visualizing data quality, as in Figure 2B. to bring the osmolarity to 300-310 mOsm as measured on an osmometer Note: Trehalose added here is separate and does not need to be considered in step 2 of the procedure.
d. If preparing more than 24 h in advance of use, filter sterilize and store at 4 °C