TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This method describes the preparation of Illumina sequencing libraries with a 1/20 scale Nextera protocol.  Typically, yeast or E. coli plasmid DNA is the input. </p>
<p> </p>
<p> </p>
<p>Method derived from: </p>
<p>Baym M, Kryazhimskiy S, Lieberman TD, Chung H, Desai MM, Kishony R (2015) Inexpensive Multiplexed Library Preparation for Megabase-Sized Genomes. PLoS ONE 10(5): e0128036. doi:10.1371/journal.pone.0128036</p>

[BEFORE_START]
<p> </p>
<p> </p>
<p>NEB buffer 4 and ATP are supplied with Exonuclease V</p>
<p> </p>
<p><a href="https://protocols.io/view/preparation-of-1-5-mg-ml-sera-mag-carbolylate-modi-g2abyae" target="_blank">Sera-mag magnetic particles</a> should be made up beforehand.  Alternatively, Agencourt AMPure XP reagent can be purchased from Beckman Coulter.</p>
<p> </p>
<p>Sage Science Pippin Prep 2% marker B and loading solution are supplied with gel cassette kit.  These reagents can also be purchased separately.</p>
<p> </p>
<p>Primer/adapter/index oligos sufficient for 384 samples are listed in the table below.  12 N7xx type and 8 S5xx type are needed for each set of 96 samples.</p>
<p> </p>
<table style="width: 473px;">
<tbody>
<tr>
<td style="width: 46px;"><strong>Index#</strong></td>
<td style="width: 413px;"><strong>   5' --&gt; 3' sequence</strong></td>
</tr>
<tr>
<td style="width: 46px;">N701</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N702</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N703</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N704</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N705</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N706</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N707</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N708</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATCCTCTCTGGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N709</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATAGCGTAGCGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N710</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N711</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N712</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N713</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATATTACAATGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N714</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATGAATGATCGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N715</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATCGATCGGTGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N716</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATAATAACGGGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N717</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATTAGAAGAAGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N718</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATGTCAGGTAGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N719</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATGCGGTCCTGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N720</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATAATCGGACGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N721</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATAACTCGTGGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N722</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATGGCCGTGGGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N723</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATTTACATGTGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">N724</td>
<td style="width: 413px;">CAAGCAGAAGACGGCATACGAGATAGTTAACAGTCTCGTGGGCTCGG</td>
</tr>
<tr>
<td style="width: 46px;">S502</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACAC CTCTCTAT TCGTCGGCAGCGTC</td>
</tr>
<tr>
<td style="width: 46px;">S503</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACAC TATCCTCT TCGTCGGCAGCGTC</td>
</tr>
<tr>
<td style="width: 46px;">S504</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACACAGAGTAGATCGTCGGCAGCGTC</td>
</tr>
<tr>
<td style="width: 46px;">S505</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACACGTAAGGAGTCGTCGGCAGCGTC</td>
</tr>
<tr>
<td style="width: 46px;">S506</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACACACTGCATATCGTCGGCAGCGTC</td>
</tr>
<tr>
<td style="width: 46px;">S507</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACACAAGGAGTATCGTCGGCAGCGTC</td>
</tr>
<tr>
<td style="width: 46px;">S508</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACACCTAAGCCTTCGTCGGCAGCGTC</td>
</tr>
<tr>
<td style="width: 46px;">S509</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACACGGCTACTCTCGTCGGCAGCGTC</td>
</tr>
<tr>
<td style="width: 46px;">S510</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACACCCTCAGACTCGTCGGCAGCGTC</td>
</tr>
<tr>
<td style="width: 46px;">S511</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACACTCCTTACGTCGTCGGCAGCGTC</td>
</tr>
<tr>
<td style="width: 46px;">S512</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACACACGCGTGGTCGTCGGCAGCGTC</td>
</tr>
<tr>
<td style="width: 46px;">S513</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACACGGAACTCCTCGTCGGCAGCGTC</td>
</tr>
<tr>
<td style="width: 46px;">S514</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACACTGGCCATGTCGTCGGCAGCGTC</td>
</tr>
<tr>
<td style="width: 46px;">S515</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACACGAGAGATTTCGTCGGCAGCGTC</td>
</tr>
<tr>
<td style="width: 46px;">S516</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACACCGCGGTTATCGTCGGCAGCGTC</td>
</tr>
<tr>
<td style="width: 46px;">S517</td>
<td style="width: 413px;">AATGATACGGCGACCACCGAGATCTACACGCGTAAGATCGTCGGCAGCGTC</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>

[GUIDELINES]
<p><strong>Materials:</strong></p>
<p><strong> </strong></p>
<p>Hard shell pcr plates (E&amp;K Scientific Framestar EK-75012 suggested) <br /> PCR film (E&amp;K Scientific T796150)</p>
<p>2 mL Deep square 96 well plates (Axygen P-2ML-SQ-C-S or equivalent)</p>
<p>8-strip PCR tube, 0.2 mL  (E&amp;K Scientific, 690030)</p>
<p>1.2 mL Deep 96 well plates (E&amp;K Scientific, OX1262 or equivalent)</p>
<p>Exonuclease V (NEB M0345L)</p>
<p>NEB Buffer 4, 10x (NEB P0756S), supplied with Exonuclease V</p>
<p>ATP 10 mM (NEB M0440S), supplied with Exonuclease V<br /> EDTA, 0.33 M<br /><a href="https://protocols.io/view/1-5-mg-ml-sera-mag-carbolylate-modified-magnetic-p-g2abyae" target="_blank">1.5 mg/mL Sera-mag magnetic particles in PEG/NaCl</a><br /> 10 mM Tris-HCl (pH 8.0 - 8.5)</p>
<p>Nextera® DNA Sample Preparation Kit, 96 samples (Illumina FC-121-1031)</p>
<p>Nextera® Index Kit, 96 indexes, 384 samples (Illumina FC-121-1012) and</p>
<p>        Additional index primer/adapter set (IDT suggested vendor, sequences listed in Before Start section)</p>
<p>KAPA HiFi Library Amplification Kit (KAPA Biosystems KK2611/KK2612)</p>
<p>Sage Science Pippin Prep gel cassette 100 - 600 bp (Sage Science CSD2010 or CDF2010)<br /> Sage Science Pippin Prep Marker B for CSD2010<br /> Sage Science Pippin Prep loading solution for CSD2010</p>
<p>Sage Science Pippin Prep Marker Mix L for CDF2010</p>
<p>Agilent High Sensitivity DNA kit (5067-4626)<br /> KAPA Illumina Platform Library quantification kit (ABI Prism) (KAPA Biosystems KK4835)</p>
<p> </p>
<p> </p>
<p><strong>Equipment:</strong></p>
<p><br /> Thermal cycler<br /> 96-well magnet plate</p>
<p>Magnet stand capable of holding 1.5 - 2 mL eppendorf tubes, such as the DynaMag-2 from Thermo-Fischer, 12321D<br /> Sage Science Pippin Prep</p>
<p>Instrument capable of small volume quantification such as a Nanodrop ND-1000 or</p>
<p>   a Qubit fluorometer <br /> Agilent Bioanalyzer<br /> qPCR instrument<br /> Illumina MiSeq</p>
<p>Illumina NextSeq<br /> High throughput robotic liquid handler (suggested)</p>
<p>Multichannel pipettes, 10 µL and 20 µL</p>
<p>96-well plate capable centrifuge</p>

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