TITLE: No Title
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[DESCRIPTION]
<p>Method used to derive single cell suspension from P1 mouse kidneys on ice, generating a cell suspension with greatly reduced artifact gene expresion changes and suitable for downstream analysis using 10x Chromium or DropSeq. </p>

[GUIDELINES]
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<p><strong>Storage Conditions of Reagents </strong></p>
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<p><strong>Reagent</strong></p>
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<p><strong>Storage Condition </strong></p>
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<p>DPBS (Thermofisher, 14190144)</p>
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<p>4°C</p>
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<p>0.5 M EDTA (Ambion, AM9260G)</p>
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<p>room temp.</p>
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<td style="width: 391px;">BSA (Sigma, A8806)</td>
<td style="width: 652px;">4°C</td>
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<p>Protease from <em>Bacillus Licheniformis </em>(Sigma, P5380)</p>
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<p>Store 100 μL aliquots (100 mg/mL) in DPBS at -80°C</p>
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<p>DNAse 1 (Applichem, A3778)</p>
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<td style="width: 652px;"> Store 10 μL aliquots (250 U/10 μL) in DPBS at -80°C</td>
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<p><strong>Required Equipment</strong></p>
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<p><strong>Equipment</strong></p>
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<p><strong>Supplier</strong></p>
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<p><strong>Catalog no.</strong></p>
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<p>gentleMACS dissociator</p>
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<p>Miltenyi</p>
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<p>130-093-235</p>
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<p><strong>The protocol workflow is as follows: </strong></p>
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<p>A. Isolate Kidney</p>
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<p>B. Initial digestion: triturate on ice</p>
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<p>C. Perform gentleMACS </p>
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<p>D. Continue triturating on ice</p>
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<p>F. Preparing cells for Chromium/DropSeq</p>
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<h3>BEFORE STARTING</h3>
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<p><strong>Prepare <em>Bacillus Licheniformis</em> enzyme mix just prior to starting dissociation:</strong></p>
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<td><strong>Volume (µl)</strong></td>
<td><strong>Reagent</strong></td>
<td><strong> Final concentration </strong></td>
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<td> 894</td>
<td> DPBS</td>
<td> 1X</td>
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<td> 1</td>
<td> 0.5 M EDTA</td>
<td> 0.5 mM</td>
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<td> 5</td>
<td> DNAse 1 (250 U/10 μL)</td>
<td> 125 U / mL</td>
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<td> 100</td>
<td> <em>B. Lich</em> (100 mg/mL)</td>
<td> 10 mg/mL</td>
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<p><strong>+25 mg tissue / 1 mL enzyme mix</strong></p>
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<p> </p>
<p>To prepare 0.01% BSA/PBS:</p>
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<p><strong>Make stock of 10% BSA in DPBS and store at -20 °C. To make PBS/BSA 0.01% aliquot 50 mL of DPBS in 50 mL conical and pipet in 50 µL of 10% BSA stock.</strong></p>
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<p><strong>Prepare 10% FBS/PBS with heat-inactivated FBS.</strong></p>
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[STEPS]
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