TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol builds on Suchan <em>et al. </em>(2016). It has been modified to work on fresh and museum samples of birds, specifically tested on Regent Honeyeater (<em>Anthochaera phrygia</em>).</p>
<p>The detailed protocol contains three main sections: (A) preparing ddRAD libraries as probes, (B) preparing whole genome libraries for samples of different quality, and (C) using probes to hybridise and isolate the same fragments from all samples.</p>
<p>Start with up to 600 ng whole genomic DNA per sample (our minimum was 14 ng), resuspended in 8 μl. Always include a negative control (containing no DNA, just buffer) in parallel to samples.</p>

[BEFORE_START]
<p>Prepare oligonucleotide working solutions and anneal adapters. Keep frozen until use.</p>
<p>All sequences are given in 5'--&gt;3' direction.</p>
<p> </p>
<p><strong>AD_SphI adapter (IS7) </strong>- used in step #3</p>
<table style="width: 406.5625px;">
<tbody>
<tr>
<td style="width: 142px; text-align: center;">
<p><strong>Name</strong></p>
</td>
<td style="width: 204.5625px; text-align: center;">
<p><strong>Sequence</strong></p>
</td>
</tr>
<tr>
<td style="width: 142px;">
<p>AD_IS7_SphI.1</p>
</td>
<td style="width: 204.5625px;">
<p>ACACTCTTTCCCTACACGACGCAT*G</p>
</td>
</tr>
<tr>
<td style="width: 142px;">
<p>AD_IS7_SphI.2</p>
</td>
<td style="width: 204.5625px;">
<p>Pho-CGTCGTGTAGGGAAAGAGTGT</p>
</td>
</tr>
</tbody>
</table>
<p><strong> </strong></p>
<p><strong>AD_MluCI adapter (IS8) </strong>- used in step #3</p>
<table style="width: 512.984375px;">
<tbody>
<tr>
<td style="text-align: center; width: 99px;">
<p><strong>Name</strong></p>
</td>
<td style="text-align: center; width: 325.984375px;">
<p><strong>Sequence</strong></p>
</td>
</tr>
<tr>
<td style="width: 99px;">
<p>AD_IS8_MluCI.1</p>
</td>
<td style="width: 325.984375px;">
<p>Pho-AATTAGATCGGAAGAGCGAGAACAA</p>
</td>
</tr>
<tr>
<td style="width: 99px;">
<p>AD_IS8_MluCI.2</p>
</td>
<td style="width: 325.984375px;">
<p>GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T</p>
</td>
</tr>
</tbody>
</table>
<p>Pho = 5' Phosphorylation</p>
<p>* = 3' phosphorothioate bond</p>
<p>These oligos are HPLC purified.</p>
<p> </p>
<p>To prepare 10 μM working solution, mix 10 μL of the RAD-P1.1 and RAD-P1.2 oligos (100 μM stock) with 80 μL of ultrapure water to obtain 100 μL of working solution. Anneal adapters as follows: heat to 95°C for 1 minute and slowly cool to 20°C at a ramp of 0.1°C/s.</p>
<p> </p>
<p><strong>ddRAD primers</strong> (10 μM) - used in step #6</p>
<table style="width: 255.609375px;">
<tbody>
<tr>
<td style="text-align: center; width: 53px;">
<p><strong>Name</strong></p>
</td>
<td style="text-align: center; width: 189.609375px;">
<p><strong>Sequence</strong></p>
</td>
</tr>
<tr>
<td style="width: 53px;">
<p>IS7</p>
</td>
<td style="width: 189.609375px;">
<p>ACACTCTTTCCCTACACGAC</p>
</td>
</tr>
<tr>
<td style="width: 53px;">
<p>IS8</p>
</td>
<td style="width: 189.609375px;">
<p>GTGACTGGAGTTCAGACGTGT</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>PE-P7-CCCCC oligo</strong> (15 μM) - used in step #17</p>
<p>ctcggcattcctgctgaaccgctcttccgatctCCCCC</p>
<p> </p>
<p><strong>P5 indexed adapters </strong>- used in step #20</p>
<table style="width: 492.03125px;">
<tbody>
<tr>
<td style="width: 192px; text-align: center;"><strong>Name</strong></td>
<td style="width: 297.03125px; text-align: center;"><strong>Sequence</strong></td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-1</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctGCAGCT</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-2</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctTCCTCC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-3</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctGAACTA</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-4</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctACAACC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-5</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctGGTAAC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-6</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctGTGGTC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-7</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctCCGCGT</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-8</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctCTGACA</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-9</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctCCGAAT</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-10</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctAGCCGC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-11</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctTAGCGC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-12</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctTGACCT</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-13</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctCTTATC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-14</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctGTAGCC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-15</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctCCATAG</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-16</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctGAGGCA</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-17</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctAATTGA</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-18</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctACTCAC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-19</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctAAGTTG</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-20</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctTACGAT</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-21</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctCACCAC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-22</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctGCATTC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-23</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctTTCCAT</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-24</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctAGGCGA</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-25</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctCTCTCG</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-26</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctGCCTTG</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-27</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctAGGTTC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-28</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctTAGCAG</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-29</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctCACTGC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-30</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctAGTGGC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-31</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctATGTAG</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-32</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctGGTACG</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-33</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctTAAGGT</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-34</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctCCTGTA</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-35</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctCCTCCA</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-36</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctACAATA</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-37</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctACTGAT</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-38</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctATGTGT</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-39</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctGTGTCC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-40</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctTAGAGT</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-41</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctGCTAAG</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-42</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctTGGACT</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-43</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctCACTTA</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-44</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctCTATTG</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-45</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctAATACA</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-46</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctATTCTT</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-47</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctGGCATC</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-48</td>
<td style="width: 297.03125px;">ctttccctacacgacgctcttccgatctGGTTGG</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_1</td>
<td style="width: 297.03125px;">AGCTGCagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_2</td>
<td style="width: 297.03125px;">GGAGGAagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_3</td>
<td style="width: 297.03125px;">TAGTTCagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_4</td>
<td style="width: 297.03125px;">GGTTGTagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_5</td>
<td style="width: 297.03125px;">GTTACCagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_6</td>
<td style="width: 297.03125px;">GACCACagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_7</td>
<td style="width: 297.03125px;">ACGCGGagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_8</td>
<td style="width: 297.03125px;">TGTCAGagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_9</td>
<td style="width: 297.03125px;">ATTCGGagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_10</td>
<td style="width: 297.03125px;">GCGGCTagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_11</td>
<td style="width: 297.03125px;">GCGCTAagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_12</td>
<td style="width: 297.03125px;">AGGTCAagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_13</td>
<td style="width: 297.03125px;">GATAAGagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_14</td>
<td style="width: 297.03125px;">GGCTACagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_15</td>
<td style="width: 297.03125px;">CTATGGagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_16</td>
<td style="width: 297.03125px;">TGCCTCagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_17</td>
<td style="width: 297.03125px;">TCAATTagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_18</td>
<td style="width: 297.03125px;">GTGAGTagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_19</td>
<td style="width: 297.03125px;">CAACTTagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_20</td>
<td style="width: 297.03125px;">ATCGTAagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_21</td>
<td style="width: 297.03125px;">GTGGTGagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_22</td>
<td style="width: 297.03125px;">GAATGCagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_23</td>
<td style="width: 297.03125px;">ATGGAAagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_24</td>
<td style="width: 297.03125px;">TCGCCTagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_25</td>
<td style="width: 297.03125px;">CGAGAGagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_26</td>
<td style="width: 297.03125px;">CAAGGCagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_27</td>
<td style="width: 297.03125px;">GAACCTagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_28</td>
<td style="width: 297.03125px;">CTGCTAagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_29</td>
<td style="width: 297.03125px;">GCAGTGagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_30</td>
<td style="width: 297.03125px;">GCCACTagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_31</td>
<td style="width: 297.03125px;">CTACATagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_32</td>
<td style="width: 297.03125px;">CGTACCagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_33</td>
<td style="width: 297.03125px;">ACCTTAagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_34</td>
<td style="width: 297.03125px;">TACAGGagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_35</td>
<td style="width: 297.03125px;">TGGAGGagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_36</td>
<td style="width: 297.03125px;">TATTGTagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_37</td>
<td style="width: 297.03125px;">ATCAGTagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_38</td>
<td style="width: 297.03125px;">ACACATagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_39</td>
<td style="width: 297.03125px;">GGACACagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_40</td>
<td style="width: 297.03125px;">ACTCTAagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_41</td>
<td style="width: 297.03125px;">CTTAGCagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_42</td>
<td style="width: 297.03125px;">AGTCCAagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_43</td>
<td style="width: 297.03125px;">TAAGTGagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_44</td>
<td style="width: 297.03125px;">CAATAGagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_45</td>
<td style="width: 297.03125px;">TGTATTagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_46</td>
<td style="width: 297.03125px;">AAGAATagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_47</td>
<td style="width: 297.03125px;">GATGCCagatcggaa</td>
</tr>
<tr>
<td style="width: 192px;">Index_P5-comp_48</td>
<td style="width: 297.03125px;">CCAACCagatcggaa</td>
</tr>
</tbody>
</table>
<p> </p>
<p>Barcoded oligonucleotides are given in capital letters.</p>
<p>These adapters are barcoded in a similar way as RAD-P1 adapters. Prepare 25 μM working solution for as many PCR wells as P1 barcoded adapters are needed. Mix 25 μl of each oligo from the pair (100 μM stock) with 50 μL of ultrapure water to obtain 100 μL working solution.</p>
<p>Anneal adaptersas follows: heat to 95°C for 1 minute and bring to 20°C with a ramp of 0.1°C/s.</p>
<p>Keep them organized in PCR strips with individually capped tubes for convenient usage. Ensure no cross-contamination of barcoded adapters when pipetting.</p>
<p> </p>
<p><strong>Indexed primers </strong>- used in step #24</p>
<table style="width: 677.1875px;">
<tbody>
<tr>
<td style="text-align: center; width: 182px;">
<p><strong>Name</strong></p>
</td>
<td style="text-align: center; width: 489.1875px;">
<p><strong>Sequence</strong></p>
</td>
</tr>
<tr>
<td style="width: 182px;">
<p>Sol-PE-PCR_F</p>
</td>
<td style="width: 489.1875px;">
<p>aatgatacggcgaccaccgagatctacactctttccctacacgacgctcttc</p>
</td>
</tr>
<tr>
<td style="width: 182px;">
<p>Index01-PE-Primer</p>
</td>
<td style="width: 489.1875px;">
<p>caagcagaagacggcatacgagatCCTGCGAcggtctcggcattcctgctgaacc</p>
</td>
</tr>
<tr>
<td style="width: 182px;">
<p>Index02-PE-Primer</p>
</td>
<td style="width: 489.1875px;">
<p>caagcagaagacggcatacgagatTGCAGAGcggtctcggcattcctgctgaacc</p>
</td>
</tr>
<tr>
<td style="width: 182px;">
<p>Index03-PE-Primer</p>
</td>
<td style="width: 489.1875px;">
<p>caagcagaagacggcatacgagatCGCATTAcggtctcggcattcctgctgaacc</p>
</td>
</tr>
<tr>
<td style="width: 182px;">
<p>Index04-PE-Primer</p>
</td>
<td style="width: 489.1875px;">
<p>caagcagaagacggcatacgagatTTGATCCcggtctcggcattcctgctgaacc</p>
</td>
</tr>
<tr>
<td style="width: 182px;">
<p>Index05-PE-Primer</p>
</td>
<td style="width: 489.1875px;">
<p>caagcagaagacggcatacgagatATCTTGCcggtctcggcattcctgctgaacc</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>Barcoded oligonucleotides are given in capital letters.</p>
<p>Each primer mix consists of the same forward (Sol-PE-PCR_F) and a plate indexed reverse primer (IndexXX-PE-Primer), each at 5 μM.</p>
<p>To prepare, mix 50 μl of the 100 μM stock primer Sol-PE-PCR_F and 50 μl primer Index01-PE-Primer (1-5) oligos with 900 μl of ultrapure water to obtain 1000 μl of a working solution.</p>
<p> </p>
<p><strong>Blocking oligos </strong>(100 μM) - used in step #26 and #29</p>
<table style="width: 766.71875px;">
<tbody>
<tr>
<td style="text-align: center; width: 95px;">
<p><strong>Name</strong></p>
</td>
<td style="text-align: center; width: 658.71875px;">
<p><strong>Sequence</strong></p>
</td>
</tr>
<tr>
<td style="width: 95px;">
<p>BO1.P5.F</p>
</td>
<td style="width: 658.71875px;">
<p>AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT</p>
</td>
</tr>
<tr>
<td style="width: 95px;">
<p>BO2.P5.R</p>
</td>
<td style="width: 658.71875px;">
<p>AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT</p>
</td>
</tr>
<tr>
<td style="width: 95px;">
<p>BO3.P7.F</p>
</td>
<td style="width: 658.71875px;">
<p>AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGIIIIIIIATCTCGTATGCCGTCTTCTGCTTG</p>
</td>
</tr>
<tr>
<td style="width: 95px;">
<p>BO4.P7.R</p>
</td>
<td style="width: 658.71875px;">
<p>CAAGCAGAAGACGGCATACGAGATIIIIIIICGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>I = Inosine</p>
<p> </p>
<p><strong>TEN Buffer </strong>(100 ml, pH 7.5)</p>
<p>1 M NaCl (5.844 g NaCl)</p>
<p>10 mM Tris-Cl + 1 mM EDTA (10 ml 10x TE)</p>
<p>Ultrapure water up to 100 ml</p>
<p> </p>
<p><strong>10% SDS</strong> (100 ml)</p>
<p>10 g SDS (mw = 288.37)</p>
<p>Ultrapure water up to 100 ml</p>
<p> </p>
<p><strong>20x SSC Buffer </strong></p>
<p>3 M NaCl (175.3 g)</p>
<p>300 mM Sodium Citrate (88.2 g)</p>
<p>pH to 7.0 with HCl</p>
<p>up to 1 L Ultrapure water</p>
<p> </p>
<p><strong>Oligo Hybridization Buffer (10x)</strong></p>
<p>500 mM NaCl</p>
<p>10 mM Tris-Cl, pH 8.0</p>
<p>1 mM EDTA, pH 8.0</p>
<p> </p>

[GUIDELINES]
<p>All purification was done using Solid Phase Reversible Immobilization (SPRI) beads with different bead : sample ratios at each step (specified in the protocol). AMPure beads are commonly used. However, we used Seramag throughout the protocol.</p>
<p>All quantitation was done by fluorescence using Qubit (Invitrogen). Fragment Analyzer, Tapestation, or LabChip GXII can also be used.</p>
<p>All visualization steps for size profiles were done using LabChip GXII (Caliper). Agarose gel, Fragment Analyzer, Tapestation, or Bioanalyzer can also be used.</p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/59mizfw.jpg" data-src="https://s3.amazonaws.com/pr-journal/59kizfw.jpg" data-ofn="hyRAD_01.jpg" /></p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/59pizfw.jpg" data-src="https://s3.amazonaws.com/pr-journal/59nizfw.jpg" data-ofn="hyRAD_02.jpg" /></p>

[STEPS]
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