TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is for performing CITE-seq only. </p>
<p> </p>
<p>Cellular Indexing of Transcriptomes and Epitopes by Sequencing (<a href="https://www.nature.com/articles/nmeth.4380" target="_blank">CITE-seq</a>) is a multimodal single cell phenotyping method developed in the <a href="http://www.nygenome.org/lab-groups-overview/technology-innovation-lab/" target="_blank">Technology Innovation lab</a> at the New York Genome Center in collaboration with the Satija lab.</p>
<p> </p>
<p><img src="https://s3.amazonaws.com/pr-journal/txwfw26.png" width="432" height="83" data-src="https://s3.amazonaws.com/pr-journal/txvfw26.png" data-ofn="Screen Shot 2018-02-26 at 5.42.11 PM.png" /></p>
<p> </p>
<p>CITE-seq uses DNA-barcoded antibodies to convert detection of proteins into a quantitative, sequenceable readout.  Antibody-bound oligos act as synthetic transcripts that are captured during most large-scale oligodT-based scRNA-seq library preparation protocols (e.g. 10x Genomics, Drop-seq, ddSeq).</p>
<p> </p>
<p><img src="https://s3.amazonaws.com/pr-journal/txyfw26.png" width="579" height="398" data-src="https://s3.amazonaws.com/pr-journal/txxfw26.png" data-ofn="Screen Shot 2018-02-26 at 5.42.29 PM.png" /></p>
<p> </p>
<p>This allows for immunophenotyping of cells with a potentially limitless number of markers and unbiased transcriptome analysis using existing single-cell sequencing approaches.</p>

[BEFORE_START]
<p>Prepare Staining buffer (2%BSA/0.02%Tween, PBS). </p>

[GUIDELINES]
<p><strong>The protocol workflow is as follows: </strong></p>
<ol>
<li>Cell staining for Drop-seq or 10x Genomics</li>
<li>​<a href="http://mccarrolllab.com/dropseq/" target="_blank">Drop-seq</a> (Macosko et al. , 2015) or <a href="https://support.10xgenomics.com/single-cell-gene-expression/library-prep/doc/user-guide-chromium-single-cell-3-reagent-kit-v2-chemistry" target="_blank">10x Genomics single cell 3’ v2 assay</a></li>
<li>cDNA amplification</li>
<li>Separating ADT-derived cDNAs (&lt;180bp) and mRNA-derived cDNAs (&gt;300bp)</li>
<li>Amplifying ADT sequencing library</li>
</ol>
<p> </p>
<p><strong>Sequencing CITE-seq libraries:</strong></p>
<p> </p>
<p style="text-align: justify;">We estimate that an average of 100 molecules per ADT per cell is sufficient to achieve useful information, we typically sequence our ADT / HTO libraries to obtain significantly more reads than this per cell. ADT and cDNA sequencing libraries can be pooled at desired proportions. We typically sequence ADT libraries in 5-10% of a lane and cDNA library fraction at 90% of a lane (HiSeq2500 Rapid Run).</p>
<p style="text-align: justify;"> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/tzkfw26.png" width="595" height="166" data-src="https://s3.amazonaws.com/pr-journal/tzjfw26.png" data-ofn="Screen Shot 2018-02-27 at 10.56.33 AM.png" /></p>
<p> </p>
<p> </p>
<p><strong>Oligonucleotide sequences: </strong></p>
<p> </p>
<p><strong>CITE-seq antibody-oligos (ADTs): </strong></p>
<p style="text-align: justify;">These contain standard small TruSeq RNA read 2 sequences and can be amplified using Illumina’s Truseq Small RNA primer sets (RPIx – primers, see example RPI1 below). See example below with a 12nt barcode:</p>
<p style="text-align: justify;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/tznfw26.png" width="606" height="20" data-src="https://s3.amazonaws.com/pr-journal/tzmfw26.png" data-ofn="Screen Shot 2018-02-27 at 10.58.09 AM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Oligos required for ADT library amplification:</strong></p>
<ul>
<li>Drop-seq P5-SMART-PCR hybrid primer (for Drop-seq only)<img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/tzqfw26.png" width="556" height="15" data-src="https://s3.amazonaws.com/pr-journal/tzpfw26.png" data-ofn="Screen Shot 2018-02-27 at 11.00.20 AM.png" /></li>
</ul>
<ul>
<li>10x Genomics SI-PCR primer (for 10x Single Cell Version 2 only)<img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/tzsfw26.png" width="572" height="16" data-src="https://s3.amazonaws.com/pr-journal/tzrfw26.png" data-ofn="Screen Shot 2018-02-27 at 11.01.15 AM.png" /></li>
</ul>
<ul>
<li>ADT cDNA PCR additive primer<img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/tzufw26.png" width="545" height="18" data-src="https://s3.amazonaws.com/pr-journal/tztfw26.png" data-ofn="Screen Shot 2018-02-27 at 11.02.13 AM.png" /></li>
</ul>
<ul>
<li>Illumina Small RNA RPI1 primer (for ADT amplification; i7 index 1, Oligonucleotide sequences © 2015 Illumina, Inc)<img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/tzwfw26.png" width="556" height="60" data-src="https://s3.amazonaws.com/pr-journal/tzvfw26.png" data-ofn="Screen Shot 2018-02-27 at 11.03.22 AM.png" /></li>
</ul>
<p> </p>

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