TITLE: No Title
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[DESCRIPTION]
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<p>Currently, most single cell protocols require the preparation of a single cell suspension from fresh tissue, a major roadblock to clinical deployment, to archived materials and to certain tissues such as adult brain. In the adult brain the harsh enzymatic dissociation harms the integrity of the cells and their RNA, and biases toward easily dissociated cell types, and is restricted to young animals.</p>
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<p>We developed DroNc-seq, a droplet microfluidic and DNA barcoding technique for analysis of RNA profiles of single nuclei from fresh, frozen or lightly fixed tissues at high throughput and low cost. The utility of DroNc-Seq lies in working with hard-to-dissociate, frozen and/or archived tissues. To demonstrate the utility of this technique, we sequenced over 39 thousand nuclei from mouse and human archived brain samples, including post-mortem human brain tissue from GTEx project.</p>
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[BEFORE_START]
<div><strong>Drop-seq Lysis Buffer</strong> (DLB6; a 10 ml stock consists of 4 ml of nuclease-free H2O, 3 ml 20% Ficoll PM‐400 (Sigma, Cat # F5415-50ML), 100 µl 20% Sarkosyl (Teknova, Inc., Cat # S3377), 400 µl 0.5M EDTA (Life Technologies), 2 ml 1M Tris pH 7.5 (Sigma), and 500 µl 1M DTT (Teknova, Inc., Cat # D9750), where the DTT is added fresh before every experiment)</div>
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[GUIDELINES]
<p>[Please note that this protocol is originally published at Protocol Exchange:</p>
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<p>Anindita Basu, Inbal Avraham-Davidi, Naomi Habib, et al. <a href="https://www.nature.com/protocolexchange/protocols/6163" target="_blank">DroNc-seq step-by-step</a>. Protocol Exchange (2017) doi:10.1038/protex.2017.094]</p>
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<p><strong>Equipment </strong></p>
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<p>a. Microfluidic chip (see CAD file). The unit in the CAD provided is 1 unit = 1 µm; channel depth on device is 75 µm.<br />b. Drop-seq microfluidic setup (see reference):<br />- optical microscope (Olympus IX83)<br />- Fast camera (Photron SA5)<br />- Three syringe pumps (KD Scientific, KDS910)<br />- Magnetic Stirrer (VP Scientific, #710D2)<br />c. Invitrogen Qubit 3.0 Fluorometer<br />d. Agilent 2100 Bioanalyzer<br />e. Illumina NextSeq 500</p>
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<div><strong>The protocol workflow is as follows:</strong></div>
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<div>1. Beads preparation</div>
<div>2. Cell culture</div>
<div>3. Tissue preservation</div>
<div>4. Nuclei isolation</div>
<div>5. Microfluidics</div>
<div>6. Droplet breakage, washes and reverse transcription (RT)</div>
<div>7. Post RT wash, exonuclease I treatment and PCR</div>
<div>8. WTA library QC and Nextera library prep</div>
<div>9. Sequencing</div>
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<div><strong>Acknowledgements:</strong></div>
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<div>We thank Rhiannon Macare, Assaf Rotem, Christoph Muus and Eugene Drokhlyansky for helpful discussions, Talia Habib for babysitting, Timothy Tickle and Asma Bankapur for technical support, Leslie Gaffney and Ania Hupalowska for help with graphics. Work was supported by Klarman Cell Observatory, NIMH grant U01MH105960, NCI grant 1R33CA202820-1 (to A.R.), and Koch Institute Support (core) grant P30-CA14051 from the NCI. Microfluidic devices were fabricated at the Center for Nanoscale Systems, Harvard University, member of NNIN, supported by NSF award no. 1541959. A.R. is an Investigator of Howard Hughes Medical Institute (HHMI). A.R. is a member of Scientific Advisory Boards for Thermo Fisher Scientific, Syros Pharmaceuticals and Driver Genomics. F.Z. is a New York Stem Cell Foundation-Robertson Investigator. F.Z. is supported by NIH through NIMH (5DP1-MH100706 and 1R01-MH110049), NSF, HHMI, the New York Stem Cell, Simons, Paul G. Allen Family, and Vallee Foundations; and James and Patricia Poitras, Robert Metcalfe, and David Cheng. D.A.W. thanks NSF DMR-1420570, NSF DMR-1310266 and NIH P01HL120839 grants for their support. NH is a HHMI fellow for Helen Hey Whitney Foundation. N.H., A.B., I.A.D., D.A.W., F.Z. and A.R. are inventors on international patent application PCT/US16/59239 filed by the Broad Institute, Harvard and MIT, relating to method of this manuscript. GTEx is supported by the Common Fund of the Office of the Director of NIH, through Contract HHSN268201000029C (to K.A LDACC, Broad Institute).</div>
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<div><strong>Corresponding authors:<br /></strong></div>
<div>Aviv Regev (aregev@broadinstitute.org) and Feng Zhang (zhang@broadinstitute.org)</div>
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<div><strong>References:</strong></div>
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<div>Macosko, E. Z. et al. Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell 161, 1202-1214, doi:10.1016/j.cell.2015.05.002 (2015).</div>
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</section>

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