TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is for performing CITE-seq and Cell Hashing in parallel. </p>
<p> </p>
<p><strong>CITE-seq: </strong></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>
<p> </p>
<p><strong>Cell Hashing:</strong></p>
<p> </p>
<p>Sample multiplexing and super-loading on single cell RNA-sequencing platforms.</p>
<p> </p>
<p><a href="https://www.biorxiv.org/content/early/2017/12/21/237693" target="_blank">Cell Hashing</a> uses a series of oligo-tagged antibodies against ubiquitously expressed surface proteins with different barcodes to uniquely label cells from distinct samples, which can be subsequently pooled in one scRNA-seq run. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its sample of origin, and robustly identify doublets originating from multiple samples.</p>
<p> </p>
<p><img src="https://s3.amazonaws.com/pr-journal/tx2fw26.png" width="578" height="235" data-src="https://s3.amazonaws.com/pr-journal/txzfw26.png" data-ofn="Screen Shot 2018-02-26 at 5.44.55 PM.png" /></p>
<p> </p>
<p> </p>

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

[GUIDELINES]
<p>For experiments involving cell hashing, we recommend using the <a href="http://satijalab.org/costpercell" target="_blank">cost per cell calculator</a> from the Satija Lab to plan experiments, determine number of hashes, number of cells to load, expected doublet rates (detected and undetected) and cost considerations. </p>
<p> </p>
<p><strong>Sequencing CITE-seq and Hashing libraries:</strong></p>
<p> </p>
<p>We estimate that an average of 100 molecules per ADT or HTO per cell is sufficient to achieve useful information, we typically sequence our ADT / HTO libraries to obtain significantly more reads than this per cell. The number of reads required to obtain 100 molecules depends on the complexity of the sequencing library (e.g. duplication rate). ADT, HTO and cDNA sequencing libraries can be pooled at desired proportions. We typically sequence ADT at 10% and HTO libraries at 5% of a lane and cDNA library fraction at 85% of a lane (HiSeq2500 Rapid Run Mode Flowcell).</p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/t2cfw26.png" width="582" height="190" data-src="https://s3.amazonaws.com/pr-journal/t2bfw26.png" data-ofn="Screen Shot 2018-02-27 at 3.01.25 PM.png" /></p>
<p> </p>
<p><strong>Oligonucleotide sequences: </strong></p>
<p> </p>
<p><strong>CITE-seq antibody-oligos (ADTs): </strong></p>
<p>CITE-seq antibody-oligos 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><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/t2efw26.png" width="566" height="17" data-src="https://s3.amazonaws.com/pr-journal/t2dfw26.png" data-ofn="Screen Shot 2018-02-27 at 3.02.35 PM.png" /></p>
<p> </p>
<p><strong>Hashtag barcoding antibody-oligos (HTOs): </strong></p>
<p>Cell Hashing antibody-oligos contain standard TruSeq DNA read 2 sequences and can be amplified using truncated versions of Illumina’s TruSeq DNA primer sets (see example D701_s below). See example below with a 12nt barcode:</p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/t2gfw26.png" width="584" height="14" data-src="https://s3.amazonaws.com/pr-journal/t2ffw26.png" data-ofn="Screen Shot 2018-02-27 at 3.04.16 PM.png" /></p>
<p> </p>
<p><strong>Oligos required for ADT and HTO 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/t2ifw26.png" width="560" height="14" data-src="https://s3.amazonaws.com/pr-journal/t2hfw26.png" data-ofn="Screen Shot 2018-02-27 at 3.06.43 PM.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/t2kfw26.png" width="587" height="17" data-src="https://s3.amazonaws.com/pr-journal/t2jfw26.png" data-ofn="Screen Shot 2018-02-27 at 3.07.34 PM.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/t2nfw26.png" width="616" height="16" data-src="https://s3.amazonaws.com/pr-journal/t2mfw26.png" data-ofn="Screen Shot 2018-02-27 at 3.08.12 PM.png" /></li>
</ul>
<ul>
<li>HTO cDNA PCR additive primer<img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/t2qfw26.png" width="593" height="16" data-src="https://s3.amazonaws.com/pr-journal/t2pfw26.png" data-ofn="Screen Shot 2018-02-27 at 3.09.05 PM.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/t2sfw26.png" width="548" height="17" data-src="https://s3.amazonaws.com/pr-journal/t2rfw26.png" data-ofn="Screen Shot 2018-02-27 at 3.09.59 PM.png" /></li>
</ul>
<ul>
<li>Illumina TruSeq D701_s primer (for HTO amplification; i7 index 1, shorter than the original Illumina sequence)<img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/t2yfw26.png" width="667" height="62" data-src="https://s3.amazonaws.com/pr-journal/t2xfw26.png" data-ofn="Screen Shot 2018-02-27 at 3.45.20 PM.png" /></li>
</ul>

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