TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Breast cancer originates in the mammary gland epithelium, however growing evidence demonstrates that the diverse array of stromal tissues influence the behavior of breast epithelium and has key roles in the pathogenesis of breast cancer. Despite increased recognition and research, the heterogeneity of the breast stroma (endothelium, fibroblasts, and adipocytes) poses challenges in elucidating which stromal populations are responsible for the complex interactions of the breast microenvironment. To this end, we have employed single cell RNA sequencing (scRNAseq) of the stromal populations within the breast. However adipose tissue, due to its delicate and lipid filled nature is not amenable to these methods of interrogation. To overcome this obstacle, we have developed a method for isolation of adipose nuclei for 10x sequencing. Together, we will use these approaches to investigate the heterogeneity of stroma and adipocytes, and determine the interactions of the breast microenvironment at single-cell resolution. </p>

[GUIDELINES]
<ol>
<li>Use of this protocol for other tissues. 
<ul>
<li>It is important to note that additional optimization is required for different tissues.</li>
<li>In particular it is important to optimize the concentration of lysing detergent (in this case NP-40) for the tissue. Also the amount of BSA in the wash buffer requires optimization. </li>
<li>Centrifugation speeds should be also be optimized. Our experience shows that nuclei after FACS purificaiton are lysed when centrifuged at 500xg. Centrifugation at slower speeds for longer periods are necessary to pool nuclei. </li>
</ul>
</li>
<li>Quality Control 
<ul>
<li>QC for nuclei can be difficult by metrics typically used for cells (ie trypan blue marks nuclei, but is used to identify dead cells). The most important is microscopy throughout the nuclei preparation to ensure that nuclei remain of high quality before proceeding with 10xseq </li>
</ul>
</li>
<li>FACS purification. 
<ul>
<li>FACS purification of nuclei after tissue preparation is paramount for successful 10x Nuclei Seq. </li>
<li>Lack of FACS purificaiton results in excessive amounts of ambient RNA that results in poor results--low UMI, falsely high number of cells (Figure 1) and poor indisitnct tSNE clustering (Figure 2) </li>
</ul>
</li>
</ol>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/658jjdn.png" data-src="https://s3.amazonaws.com/pr-journal/658jjdn.png" data-ofn="Screen Shot 2018-09-10 at 4.58.32 PM.png" /></p>
<p><strong>Figure 1--Ambient RNA resulsts in false positive cells</strong>: Only 10K nuclei were loaded. But 82K nuclei were detected, also a majority of 'cells' head low UMI and gene counts. </p>
<p> </p>
<p> </p>
<p><img src="https://s3.amazonaws.com/pr-journal/659jjdn.png" data-src="https://s3.amazonaws.com/pr-journal/659jjdn.png" data-ofn="Screen Shot 2018-09-10 at 4.58.10 PM.png" /></p>
<p><strong>Figure 2--Ambient RNA results in poor clustering</strong>: ambient RNA from nuclei that were not FACS purified results in tSNE clustering where marker genes for various cells types are found in all cells.  </p>

[STEPS]
SECTION: Prepare Materials
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SECTION: Prepare Tissue for Dounce Homogenization
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SECTION: Isolate Nuclei
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SECTION: Wash and Stain Nuclei
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SECTION: FACS Purification of Nuclei
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SECTION: Post-FACS Preparation of Nuclei for 10xSeq
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