TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div title="Page 1">
<div>
<div>
<div>
<p>We have continued to optimize this protocol since doing the experiments in the<a href="http://www.sciencedirect.com/science/article/pii/S0092867415005498" target="_blank"> Macosko <em>et al. Cell</em> paper</a>. What we are sharing here is our current, optimized protocol. As a result, this protocol will not precisely match the methods section of the paper, which is a description of the experiments done in the paper. Please feel free to use these optimizations without author consideration - just acknowledge “helpful advice from Evan Macosko, Melissa Goldman and Steve McCarroll”, and please also include the URL and version number of the protocol in your methods section. This protocol also includes hints, suggestions and images that we could not fit into the the methods section of the paper.</p>
</div>
</div>
</div>
</div>

[GUIDELINES]
<div title="Page 1">
<p style="text-align: justify;">Starting with a species-mixing experiment is a critical first step for validating that your experimental setup is successfully producing libraries with high single-cell integrity. <em>It is critical to do a successful species-mixing experiment before doing any other single-cell experiment</em>. The protocol here includes step-by-step instructions for performing Drop-seq analysis of a human and mouse cell mixture (HEK and 3T3 cells). Successful demonstration that cell barcodes yield organism-specific libraries in such an experiment will ensure that you have all the experimental and computational components in place to produce high-quality single-cell libraries.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>The protocol workflow is as follows: </strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">1. Pre-run setup (Steps 1-9)</p>
<p style="text-align: justify;">2. Loading cells and beads (Steps 10-14)</p>
<p style="text-align: justify;">3. Flow rates (Step 15)</p>
<p style="text-align: justify;">4. Starting your run (Steps 16-18)</p>
<p style="text-align: justify;">5. Assessment of droplet quality and bead doublets (Steps 19-21)</p>
<p style="text-align: justify;">6. Breakage (Steps 22-31)</p>
<p style="text-align: justify;">7. Reverse transcription (Steps 32-36)</p>
<p style="text-align: justify;">8. Exonuclease I treatment (Steps 37-40)</p>
<p style="text-align: justify;">9. Preparing for PCR (Steps 41-46)</p>
<div title="Page 16">
<p>10. PCR program (Step 47)</p>
<p>11. Purification of the cDNA library and analysis on the BioAnalyzer (Steps 48-52)</p>
</div>
<p style="text-align: justify;">12. Tagmentation of cDNA with Nextera XT (Steps 53-60)</p>
<p style="text-align: justify;">13. Purification of the tagmented library and analysis on the BioAnalyzer (Steps 61-65)</p>
<p style="text-align: justify;">14. Sequencing your sample (Steps 66-67)</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Videos and FAQs</strong></p>
<p style="text-align: justify;"> </p>
<p>These <a href="http://mccarrolllab.com/drop-seq-troubleshootingtutorial-with-pictures-videos/" target="_blank">tutorials, images and diagrams</a> may be helpful in building your own Drop-seq setup and doing Drop-seq experiments in your lab.</p>
<p>We have also created a YouTube channel with a variety of <a href="https://www.youtube.com/channel/UCRptwggZzFyM51R5iAI-07Q" target="_blank">videos</a> to help scientists through the steps that most benefit from watching.</p>
<p>This <a href="http://mccarrolllab.com/drop-seq-faq/" target="_blank">FAQ</a> provides also provides supplementary information.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Materials </strong></p>
<p style="text-align: justify;"> </p>
<ul style="text-align: justify;">
<li><strong>Barcoded beads</strong>.  In order to have a scalable, commercial solution available for users, we taught scientists at a local company (Chemgenes) the split-pool oligo synthesis process we describe in the <em>Cell</em> paper.  We have extensively tested these beads in Drop-seq.  We have asked Chemgenes to make these beads available directly to anyone who orders them. The beads have catalog number CSO-2011.</li>
<li><strong>Microfluidic devices</strong>
<ul>
<li>Here you can download the <a href="http://mccarrolllab.com/wp-content/uploads/2015/05/CADfile_FINAL.dwg" target="_blank">CAD file</a> for the co-flow microfluidics device, which was designed by our colleague Anindita Basu in the labs of Aviv Regev and David Weitz.  This is a passive-flow PDMS device that can be synthesized in any academic or commercial microfluidics facility.</li>
<li>Alternatively, microfluidic devices can be purchased ready-made from two companies, Nanoshift and FlowJEM.  Simply tell them you’re interested in ordering the latest Drop-seq devices for a quote.  See our <a href="http://mccarrolllab.com/drop-seq-faq/" target="_blank">FAQ</a> page for more information.</li>
</ul>
</li>
</ul>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Software </strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">Download here our latest cookbook for core computational analysis of Drop-seq data, which shows you how to transform raw sequence data into an expression measurement for each gene in each individual cell (<a href="http://mccarrolllab.com/wp-content/uploads/2016/03/Drop-seqAlignmentCookbookv1.2Jan2016.pdf" target="_blank">Drop-seqAlignmentCookbookv1.2Jan2016</a>).  The software was developed by Jim Nemesh in our lab, and the current implementation can be found here (<a href="http://mccarrolllab.com/download/1276/" target="_blank"> Drop-seq_tools-1.13.zip (2380 downloads) </a>).</p>
<p style="text-align: justify;">We add updates (and revise the protocol date) whenever we see opportunities to make something better, faster or more clear.  We will increment the version number whenever we revise the software or change something substantive about the recommended series of steps.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/r9mfw26.png" width="628" height="211" data-src="https://s3.amazonaws.com/pr-journal/r9kfw26.png" data-ofn="Screen Shot 2018-01-13 at 12.52.51 PM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">We further analyze the resulting data using our colleague <a href="http://satijalab.org/" target="_blank">Rahul Satija</a>‘s fantastic single-cell analysis package <a href="http://www.satijalab.org/seurat.html" target="_blank">Seurat</a>, which produced all of the clustering analyses in the <em>Cell</em> paper.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Data resources</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">Here are alignment data resources (the same ones as described in our paper) for<a href="ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE63nnn/GSE63269/suppl/GSE63269_hg19_mm10_transgenes_reference_metadata.tar.gz" target="_blank"> </a><a href="ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE63nnn/GSE63472/suppl/GSE63472_mm10_reference_metadata.tar.gz" target="_blank">MOUSE, </a><a href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1629193" target="_blank">HUMAN</a>, and <a href="ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE63nnn/GSE63269/suppl/GSE63269_hg19_mm10_transgenes_reference_metadata.tar.gz" target="_blank">MIXED</a> (human+mouse).  Scientists in the Drop-seq community have also now created such resources for many other organisms.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Example data sets</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">We have deposited all of the raw and processed data sets from the Drop-seq experiments described in the <em>Cell</em> paper (Macosko et al, 2015) in Gene Expression Omnibus, accession <a href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63473" target="_blank">GSE63473</a>.  Cluster assignments for the 44,808 retinal cells in the paper are <a href="http://mccarrolllab.com/wp-content/uploads/2015/05/retina_clusteridentities.txt" target="_blank">here</a>.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Necessary start-up equipment:</strong></p>
<div title="Page 2">
<p style="text-align: justify;"> </p>
<div title="Page 2">
<ul>
<li style="text-align: justify;">
<p><strong>An inverted microscope</strong> to view the device (we use the Motic AE31)</p>
</li>
<li style="text-align: justify;">
<p><strong>Three syringe pumps</strong> (we use KD Scientific Legato 100)</p>
</li>
<li style="text-align: justify;">
<p><strong>A highly controllable, powerful magnetic mixing system</strong>; I have looked at options extensively and the VP Scientific (Part #710D2) magnetic stirrer is both sufficiently powerful and gentle to keep the beads suspended while not breaking them. You may consider buying a second mixer if you think it will be necessary to also mix your cells while they are sitting in the syringe, but typically we do not mix our cells. We load one <strong>mixing disc</strong> into the beads syringe to stir the beads, which you can also get from VP (VP cat # 772DP-N42-5-2). These mixing discs are thicker than the ones we had recommended previously, and therefore are better at keeping the beads uniformly suspended.</p>
</li>
<li style="text-align: justify;">
<p><strong>3 mL syringes</strong> (we use BD #309657)</p>
</li>
<li style="text-align: justify;">
<p><strong>Tubing to connect syringes to the device</strong>: Scientific Commodities, inc. (cat # BB31695-PE/2)</p>
</li>
<li style="text-align: justify;">
<p><strong>Luer lock 2 6-gage needles</strong>. The connection between the tubing and the needle is intentionally quite snug; I do not use 27G needles (though the fit is easier) because the beads are more likely to clog in the smaller bore.</p>
</li>
<li style="text-align: justify;">
<p><strong>PDMS co-flow microfluidic droplet generation device</strong>. We provide a CAD file with the <em>Cell</em> paper for the devices we used for all experiments in the paper. These devices were designed by our collaborator Anindita (Oni) Basu. Though many ideas and much optimization went into their design, their construction is straightforwardly accomplished in any academic or commercial microfluidics facility, as they are passive PDMS devices. Some commercial microfluidics companies that will make custom devices from CAD files include FlowJem, Nanoshift, and Dolomite Microfluidics.</p>
</li>
<li style="text-align: justify;">
<p><strong>100 micron cell strainers</strong> for beads (VWR cat #21008-950)</p>
</li>
<li style="text-align: justify;">
<p><strong>40 micron cell strainers</strong> for cells (VWR cat #21008-949)</p>
</li>
<li>
<p style="text-align: justify;"><strong>Fuchs-Rosenthal hemocytometer</strong> ( Incyto # DHC-F01)</p>
</li>
</ul>
<p> </p>
<p><strong>Required Primers: </strong></p>
<p> </p>
<p><strong><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/r9cfw26.png" width="561" height="278" data-src="https://s3.amazonaws.com/pr-journal/r9bfw26.png" data-ofn="Screen Shot 2018-01-13 at 10.46.27 AM.png" /></strong></p>
<p> </p>
<div title="Page 3">
<p> </p>
<p style="text-align: justify;"><strong>Ordering the beads and what to do when they arrive:</strong></p>
<p style="text-align: justify;"> </p>
<div title="Page 3">
<p style="text-align: justify;">We taught scientists at a local company, Chemgenes, how to do the split-and-pool synthesis described in the Cell paper. We have extensively tested these beads and in fact used them for all the experiments in the paper. Chemgenes (<a href="http://www.chemgenes.com" target="_blank">http://www.chemgenes.com</a>) has made these “Barcoded Bead SeqB” beads available for purchase (you can ask them for the beads used in Macosko et al.)</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">The beads arrive as a dry resin. Wash the resin once with 30 mL ethanol, then twice with 30 mL TE-TW. Resuspend in 20 mL TE-TW, pass through a 100 micron strainer, and count the beads using a Fuchs-Rosenthal hemocytometer. Store the counted beads at 4 C. We have stored beads in this way for &gt;6 months without any apparent loss of activity.</p>
<p style="text-align: justify;"> </p>
<div title="Page 3">
<p style="text-align: justify;"><em>Note: to pellet the beads when washing, we centrifuge at 1000xg for 1 min. Your centrifuge may require more time, or an adjustment of brake speed, which can kick up beads from the bottom if it is too high.</em></p>
<p style="text-align: justify;"> </p>
<div title="Page 4">
<p style="text-align: justify;"><strong>Arranging and connecting your droplet generation set-up:</strong></p>
<p style="text-align: justify;"> </p>
<div title="Page 4">
<p style="text-align: justify;">Set up the three syringe pumps next to the inverted microscope. It is best to have the bead pump resting on its side so that the syringe is angled down (rather than positioned horizontally, see figure below). We accomplish this by resting the bead pump on a shelf above the microscope. Your magnetic stirrer should be positioned close to the barrel of the bead syringe; once you find a good angle to get good mixing in the syringe you can fix the magnet in place, although it can be helpful to be able to move the magnet away from the syringe as the volume decreases in order to prevent the mixing disc from getting stuck in a vertical position.</p>
<p> </p>
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/r9dfw26.png" width="399" height="510" data-src="https://s3.amazonaws.com/pr-journal/r9dfw26.png" data-ofn="Screen Shot 2018-01-13 at 10.49.35 AM.png" /></p>
<p style="text-align: center;"> </p>
<div title="Page 5">
<div>
<p><strong>Chemical reagents:</strong></p>
<p> </p>
<p><strong>For cells</strong></p>
<p>●  TrypLE Express Enzyme (Life Technologies, #12604013)</p>
<p>●  <strong>BSA</strong>: make a 10% solution using BSA powder (Sigma #A8806), store aliquots at -20 C</p>
</div>
<div>●  <strong>PBS-BSA</strong>: make this fresh before each experiment</div>
<div>○ 1XPBS
<p>○ 0.01% BSA (use the 10% stock)</p>
<p> </p>
<div title="Page 5">
<p><strong>For beads</strong></p>
<p>● <strong>Lysis Buffer (makes 1 mL)</strong>: can store large stocks without DTT at room temperature</p>
<p>○  500 ul H2O</p>
<p>○  300 ul 20% Ficoll PM-400 (GE Healthcare)</p>
<p>○  10 ul 20% Sarkosyl (Sigma #L7414))</p>
<p>○  40 ul 0.5 M EDTA (Life Technologies)</p>
<p>○  100 ul 2 M Tris pH 7.5 (Sigma)</p>
<p>○  50 ul 1 M DTT → add this just prior to starting each Drop-seq experiment</p>
<p> </p>
<p><strong>Droplet generation oil</strong></p>
<div title="Page 5">
<p>● Bio-Rad, catalog # 186-4006</p>
<p> </p>
<p><strong>Post droplet generation</strong></p>
<div title="Page 5">
<div>
<p>●  <strong>6XSSC</strong></p>
<p>● <strong> Perfluorooctanol (PFO)</strong> ( Sigma #370533)</p>
<p style="text-align: justify;">●  <strong>TE-SDS (makes 50 mL)</strong>:</p>
<p style="text-align: justify;">○ 10mMTrispH8.0+1mMEDTA</p>
<p style="text-align: justify;">○ .5% SDS</p>
<p style="text-align: justify;">● <strong>TE-TW solution</strong>:<br />○ 10mMTrispH8.0+1mMEDTA</p>
<p style="text-align: justify;">○ 0.01% Tween-20</p>
<p style="text-align: justify;">● <strong>10 mM Tris pH 8.0</strong></p>
</div>
<div style="text-align: justify;">
<div>
<p> </p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
<p style="text-align: justify;"><strong><em> </em></strong></p>
<p style="text-align: justify;"><strong>Your first Drop-seq run:</strong></p>
<p style="text-align: justify;"> </p>
<div style="text-align: justify;" title="Page 6">
<div>
<div>
<p><strong>The first experiment you should perform in your lab is</strong> <strong>with a mixture of intact human (HEK) and mouse (3T3) cells as we did in the <em>Cell</em> paper.</strong> Using a mixture of human and mouse cells will allow you to evaluate whether the loading concentration, droplet quality, and downstream library preparation are all working to give you data that is single-cell-resolution. It will also allow you to measure the single-cell purity of your libraries and your cell doublet rate. <strong>We cannot emphasize strongly enough that it is critical to start with species-mixing experiments in order to evaluate the quality of the data you are generating and know whether it is truly single-cell.</strong></p>
<p><strong> </strong></p>
<p>Using HEK and 3T3 cells (rather than a different mouse and human cell line) will also allow you to compare your transcript yield to the data in Macosko <em>et al</em>. 2 015, to evaluate sensitivity (capture rates).</p>
<p> </p>
<p>Once you have been successful, please email us a plot of your species-mixing results analogous to the plot in Figure 3A of the <em>Cell</em> paper. We would love to keep a gallery of people’s successful Barnyard plots (and, with permission, to post it online). You can email us at dropseq@gmail.com .</p>
</div>
</div>
</div>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<div style="text-align: justify;" title="Page 6">
<div>
<div>
<div>
<p><strong>Anticipating your yield of STAMPs</strong></p>
<p> </p>
<p>The number of STAMPs generated per hour is a product of three factors:</p>
</div>
</div>
</div>
</div>
<div title="Page 6"> </div>
<div title="Page 6"><span style="text-align: justify;">1. The concentration of cells used. A higher concentration of cells yields higher </span>throughput,<span style="text-align: justify;"> but also introduces more doublets and impurities (see Figure S3B in Macosko et al. 2015)</span></div>
<div title="Page 6"><br /><span style="text-align: justify;">2. The concentration of beads used. We keep this fixed at around 120 beads/ul, which generates fewer than 5% bead doublets.</span></div>
<div title="Page 6"> </div>
<div title="Page 6"><span style="text-align: justify;">3. The size of droplets used. For the <em>Cell</em> paper, we used a microfluidic device that generates droplets that are 125-microns in diameter (almost exactly 1 </span>nL<span style="text-align: justify;">). However, since the paper’s publication, we have learned from our own experience, and from that of colleagues, that home-made microfluidic devices can have significant variations in droplet diameter, based on several factors related to microfluidic device master fabrication. </span><em style="text-align: justify;"><strong>We therefore recommend that you perform a test of droplet volume for devices fabricated with a particular master (the procedure for measuring droplet volume can be found at the end of this protocol)</strong></em><span style="text-align: justify;">. Please also note that if your droplet size differs significantly from 125-microns, you may need to adjust your flow rates from the numbers recommended in this protocol. We have used devices that produce as small as </span>90 micron<span style="text-align: justify;"> droplets without observing any major change in Drop-seq data quality. We anticipate that droplet sizes in the range of 90-140 microns in diameter should produce perfectly fine data.</span><br />
<div title="Page 7">
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">We recommend starting with a cell concentration of 100 cells/ul (final concentration in droplets will be 50 cells / ul when mixed 1:1 with lysis buffer and beads). At this concentration, ~5% of beads collected will have been exposed to a cell (a.k.a. STAMPs). If beads are flowed in at a concentration of 120 beads/ul, this yields 30,000 STAMPs (4 mL/hr * 120000 beads/mL * 0.05 = 24,000). Only 20-40% of beads are recovered after all washing and enzymatic steps, meaning that, practically speaking, ~10,000 STAMPs can be generated in 1-2 hours of droplet generation.</p>
<p style="text-align: justify;"> </p>
<div title="Page 19">
<div>
<div>
<p> </p>
<p style="text-align: justify;"><strong>Troubleshooting:</strong></p>
<p style="text-align: justify;"><strong> </strong></p>
<p style="text-align: justify;"><strong>Low-quality droplets</strong></p>
<p style="text-align: justify;"> </p>
</div>
</div>
<div>
<p style="text-align: justify;">From time to time, we experience runs where droplet size is not uniform. We have identified three potential causes:</p>
<div title="Page 19"> </div>
<div style="text-align: justify;" title="Page 19"><strong style="text-align: justify;">1. Failure to wait long enough for the flow to stabilize.</strong><span style="text-align: justify;"> It takes time to push all the destabilized flow through the outflow tube. You may need to wait longer to avoid having this volume in your sample.</span></div>
<div style="text-align: justify;" title="Page 19"><br /><strong style="text-align: justify;">2. Device imperfections.</strong><span style="text-align: justify;"> We generally use around 4,000 ul/hr as our flow rate for the cell and bead channels. However, we have seen batch-to-batch variation in droplet quality produced from individual devices. If you notice droplet quality problems, reduce the flow rate of cells and beads to 3,500 ul/hr, and decrease the oil flow rate proportionally. If this still does not produce good-quality droplets, consider switching to a new device.</span></div>
<div style="text-align: justify;" title="Page 19"><br /><strong style="text-align: justify;">3. Devices are old (&gt;3 months).</strong><span style="text-align: justify;"> Over time, our experience is that the Aquapel coating somehow degrades, which causes droplet quality to also decline. You may need fresh devices, or to re-treat your old devices with Aquapel.</span></div>
<div style="text-align: justify;" title="Page 19"><br /><strong style="text-align: justify;">4. Air bubbles in the microfluidic device.</strong><span style="text-align: justify;"> If you observed air bubbles accumulating in the microfluidic channels during droplet generation, this likely explains poor emulsion quality. Avoid storing cell suspension buffers on ice--when the buffer is brought up to room temperature, it can release gas (gas is more soluble in liquids at lower temperatures).</span><br />
<div title="Page 19">
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Beads breaking during droplet formation</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">It is important to mix the beads to prevent them from settling; however, overly vigorous mixing will shear the beads. <strong>We strongly recommend using the V&amp;P Scientific Mixing System described in the protocol, </strong>at the speeds described. A small amount of bead fragmentation during droplet generation is expected, but these fragments should be nearly absent from the bead pool once you are counting beads for PCR amplification.</p>
<p style="text-align: justify;"> </p>
<div title="Page 19">
<p style="text-align: justify;"><strong>Droplets break poorly/precipitate at oil-aqueous interface</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">The amount of protein in your cell suspension strongly influences the “cleanliness” of the oil-aqueous interface after droplet breakage. For example, a lot of BSA (e.x. 0.2%) can produce a snow-like precipitate (though it does not appear to affect the library quality).<strong> Serum is strongly inhibitory to Drop-seq and must be washed out completely before running cells.</strong> Serum will also produce a lot of precipitate at the oil-aqueous interface.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Questions </strong></p>
<p style="text-align: justify;"> </p>
<p>Please email questions to dropseq@gmail.com. We’ll do our best to answer them!</p>
<p> </p>
<p><em>Melissa Goldman, Jim Nemesh, Evan Macosko, Alec Wysoker, Steve McCarroll</em></p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.