The authors would like to thank members of the Yi laboratory especially Zhaojie Zhang for fruitful discussions regarding linker design and ligation efficiencies, as well as the American Cancer Society for supporting this work through a postdoctoral fellowship (#125209) to J.E.L. Research reported in this publication was also supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01AR059697 (to R.Y.) and a research grant from the Linda Crnic Institute for Down Syndrome. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

NOTE: It is critical to maintain RNase-free conditions during the entire procedure.
1. Adenylation of 3&#8217; Linker
<ol>
	<li>Dilute DNA oligonucleotide designed for 3&#8217; ligation reactions to 100 &#956;M in nuclease-free H2O. 
	NOTE: The oligonucleotide should have a 5&#8217; phosphate group, a randomized dinucleotide at the 5&#8217; end, and a 3&#8217; dideoxycytosine. The 5&#8217; phosphate group is a requirement of the Mth enzyme for efficient 5&#8217; adenylation10. The phosphate can be added by pre-treatment of the oligonucleotide with a polynucleotide kinase, or ordered with the modification directly. The randomized dinucleotide at the 5&#8217; end is important for minimizing the sequence preference which RNA ligases exhibit for certain end-end joining reactions over others6. The 3&#8217; dideoxycytosine places a blocking element at the 3&#8217; end of the oligonucleotide to inhibit the formation of linker-linker concatamers during subsequent ligation steps and also serves to block the 3&#8217; end of the miRNA-Linker molecule formed at the end of the 3&#8217; ligation reaction11. The oligonucleotide linker sequence chosen for the unbiased miR-Seq method is as follows: 5&#8217;PO4NNTGGAATTCTCGGGTGCCAAGG-dideoxyC3&#8217;.</li>
	<li>Assemble the following adenylation reaction: 200 pmol 3&#8217; linker oligonucleotide, 4 &#956;l 10x&#160;5&#8217; DNA adenylation buffer, 4 &#956;l 1 mM ATP, 4 &#956;l Mth RNA ligase, nuclease-free H2O to a final volume of 40 &#956;l.</li>
	<li>Incubate reaction at 65 &#176;C for 1 hr. Terminate reaction by heating to 85 &#176;C for 5 min.</li>
	<li>Precipitate adenylated linker by the addition of 2.5 volumes of 100% ethanol and 1/3 volume of 10 M ammonium acetate to remove residual ATP and to prevent unanticipated ligations in future reactions.
	<ol>
		<li>Briefly, mix the alcohol, salt, and oligo solution to homogeneity, spinning at full speed in a microcentrifuge (~15,000 x g) at 4 &#176;C for 20 min. Wash the pellet in 70% ethanol, air dry, and resuspend in the appropriate volume of nuclease-free H2O to yield 50-100 &#956;M of adenylated oligo.</li>
	</ol>
	</li>
	<li>Confirm adenylation by running an 18% polyacrylamide gel.
	<ol>
		<li>Prepare the gel by mixing the following together: 6.3 g urea, 6.75 ml of 40% acrylamide, and 1.5 ml of 10x&#160;TBE. Add H2O to 15 ml.</li>
		<li>Mix until urea is completely dissolved and add 150 &#181;l 10% (w/v) ammonium persulfate (APS) and 15 &#181;l tetramethlyethylenediamine (TEMED).</li>
		<li>Once gel has set, pre-run for 30 min at 24 V/cm of gel width with 1x&#160;TBE running buffer at room temperature. Once 30 min is up flush wells with running buffer.</li>
		<li>Mix 5 pmol of adenylated oligonucleotides with and equal volume of 2x&#160;denaturing RNA loading buffer (95% (v/v) Formamide, 0.02% (w/v) SDS, 0.02% bromophenol blue (w/v),0.01% (w/v) Xylene Cyanol, 1.0 mM EDTA), heat briefly to 75 &#176;C, and snap cooled on ice. Dispense entire sample into the well of the gel. Also include an identical amount of non-adenylated control, and low molecular weight size standards.</li>
		<li>Run the gel at 24 V/cm until the bromophenol blue is &#190; of the way to the bottom of the gel. Disassemble apparatus, post-stain gel with 1x&#160;sybr-gold solution in 1x&#160;TBE and visualize on UV-transilluminator box (Figure 2). 
		NOTE: Gel purify the adenylated oligo in a manner similar to that described above if &#8220;no input RNA&#8221; controls are resulting in a high amount of PCR product as compared to samples with input RNA.</li>
	</ol>
	</li>
</ol>
2. Linker Ligation to 3&#8217; End of miRNA
<ol>
	<li>3&#8217; Ligation
	<ol>
		<li>Assemble the following components on ice in the order listed: 1.0 &#956;l 50% (w/v) PEG 8000, 0.5 &#956;l 10x&#160;RNA ligase buffer (500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 10 mM DTT), 1.0 &#956;l adenylated 3&#8217; Linker (100 &#956;M), 0.5 &#956;l RNase inhibitor, 0.5-8.0 &#956;g total RNA, 0.5 &#956;l T4 RNA ligase 2, nuclease-free H2O to a final volume of 5 &#956;l.</li>
		<li>Mix the reaction by pipetting as the solution is too viscous to vortex due to the high concentration of PEG 8000. Once thoroughly mixed, place the reaction in a thermal cycler with a heated lid. Set the block to 16 &#176;C, and incubate for 4 hr. 
		NOTE: The reaction may be scaled up to a final volume of 20 &#956;l without any loss in ligation efficiency. React for 4 hr&#160;at ambient temperature or overnight at 4 &#176;C to yield essentially identical ligation efficiencies.</li>
	</ol>
	</li>
	<li>Gel Purification
	<ol>
		<li>Following 3&#8217; ligation, mix the reaction with an equal volume of 2x&#160;RNA loading buffer. Heat to 70 &#176;C for 5 min, and snap cool on ice.</li>
		<li>Run the sample on a 15% polyacrylamide gel for PAGE purification.
		<ol>
			<li>Prepare a 15% polyacrylamide gel containing 7 M urea, in a fashion similar to that described previously in step 1.5.</li>
			<li>Pre-run the gel at 24 V/cm of gel width for at least 30 min. Turn off power supply, flush wells, load sample and run gel at same voltage as pre-run.</li>
			<li>Load the sample and run the PAGE until the bromophenol blue has just exited the gel. The desired product will migrate close to the xylene cyanol.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Staining, Visualization, and Excision
	<ol>
		<li>Disassemble gel apparatus and place gel in clean tray with 1x&#160;running buffer (TBE), 1x&#160;sybr-gold, and rock for 15 min.</li>
		<li>Remove gel from staining dish and visualize on UV transilluminator box covered with clean plastic wrap. The ligation product should be around 45 nucleotides in length.</li>
		<li>Excise the region containing the ligation product with a clean razor blade and place in 0.5 ml micro-centrifuge tube.</li>
	</ol>
	</li>
	<li>Product Isolation and Precipitation
	<ol>
		<li>Pierce bottom of 0.5 ml microcentrifuge tube with a 30 G&#160;needle and place the pierced tube in a second clean, low-retention, 1.5 ml microcentrifuge tube. Centrifuge for 5 min at ~15,000 x g.</li>
		<li>Visually confirm that the entire gel slice has moved through the hole in the inner tube. If necessary, use a clean pipette tip to push some gel fragments closer to the hole.</li>
		<li>Discard the empty 0.5 ml tube and add 400 &#956;l of high-salt column buffer (HSCB 400 mM NaCl, 25 mM Tris-HCl pH 7.5, 0.1% SDS) to the acrylamide slurry. Rock tube(s) at 4 &#176;C overnight.</li>
		<li>Transfer slurry to a 0.22 micron cellulose acetate spin column using a wide-bore pipette tip. Centrifuge for 3 min at room temperature at ~15,000 x g to collect all liquid away from acrylamide gel matrix.</li>
		<li>Transfer the liquid to a clean, low-retention, 1.5 ml tube containing 1 &#956;l of 20 mg/ml glycogen. Add an equal volume of isopropanol and 1/10th volume sodium acetate. Mix solution by inverting tube several times and precipitate in -80 &#176;C freezer for 20 min.</li>
		<li>Centrifuge at full speed at 4 &#176;C for 20 min to pellet the nucleic acid. Remove supernatant and wash in 70% ethanol. Air dry pellet for 10 min at room temperature and proceed directly to 5&#8217; ligation step.</li>
	</ol>
	</li>
</ol>
3. Linker Ligation to 5&#8217; End of miRNA-3&#8217; Linker Hybrid
<ol>
	<li>To pelleted nucleic acid, add the following components: 2 &#181;l PEG 8000 (50% w/v), 0.5 &#181;l 10x&#160;RNA Ligase Buffer, 0.5 &#181;l ATP (10 mM), 0.5 &#181;l NN-RNA oligo (100 &#181;M), and H2O to a final volume of 4 &#181;l. 
	NOTE: The composition of the &#8216;NN-RNA oligo&#8217; is as follows 5&#8217;-inverted dideoxy-TCUACAGUCCGACGAUCNN3&#8217; and was purified via HPLC by the manufacturer.</li>
	<li>Mix this sample by pipetting up and down repeatedly. Do this for some time (up to 5 min) to ensure that the pellet has been completely re-solubilized. If re-solubilization is problematic, heat the sample briefly (3-5 min) to 37 &#176;C and resume pipetting to mix.</li>
	<li>Once the pellet is back in solution, transfer the contents to a clean PCR tube containing 1 &#956;l of a 1:1 mixture of RNase inhibitor and T4 RNA Ligase 1 (5 units). Again mix by pipetting up and down.</li>
	<li>Incubate the reaction in a thermal cycler at 37 &#176;C for 4 hr. Proceed immediately to cDNA synthesis.</li>
</ol>
4. Reverse Transcription/cDNA Synthesis
<ol>
	<li>Add the following components to the 5&#8217; Ligation reaction: 2 &#181;l 5x&#160;RT buffer, 0.75 &#181;l 100 mM DTT, 1 &#181;l Illumina RT Primer 5&#8217;-GCCTTGGCACCCGAGAATTCCA-3&#8217; (1 &#181;M), and 0.5 &#181;l dNTPs (10 mM).</li>
	<li>Mix by pipetting and heat to 65 &#176;C for 5 min. Then slowly cool on the bench top.</li>
	<li>Once reaction has cooled to room temperature, place tube on ice and add 0.5 &#956;l each of reverse transcriptase enzyme and RNase inhibitor. Mix by pipetting and place in thermal cycler for the extension step indicated in the manufacturer&#8217;s instructions.</li>
	<li>Once complete add 5 &#956;l of nuclease-free H2O to bring the final volume of the cDNA library up to 15 &#956;l.</li>
</ol>
5. Library Preparation
<ol>
	<li>For the indexing PCR, assemble the following reaction on ice: 6 &#181;l 5x&#160;Phusion Buffer, 1 &#181;l dNTPs (10 mM), 0.9 &#181;l&#160;DMSO, 0.75 &#181;l 5&#8217; Primer (25 &#181;M), 0.75 &#181;l 3&#8217; Primer (25 &#181;M), 3 &#181;l cDNA library (variable concentration depending on the amount of the input), 0.5 &#181;l Phusion Polymerase (1 unit), nuclease-free H2O to a final volume of 30 &#956;l. Choose the primer sequences to assure compatibility with the template and the sequencing platform of choice.
	<ol>
		<li>Run the PCR reaction with the following settings: initial denaturation at 98 &#176;C for 3 min, for the first 4 cycles, denature 98 &#176;C for 15 sec, anneal at 50 &#176;C for 15 sec, extend at 72 &#176;C for 15 sec. 
		NOTE: Depending on the amount of the input material, vary the total PCR cycle numbers. Typically, for an input of 1-2 &#181;g of total RNA, 13 total PCR cycles are sufficient to generate the miR-Seq library.</li>
		<li>Carry out subsequent cycles, e.g., cycles 5-12, in a two-step manner where the annealing and extension cycles are combined and carried out at 70 &#176;C for 30 sec&#160;and the denaturation is again 98 &#176;C for 15 sec. 
		NOTE: The reaction volume is 30 &#956;l to permit pausing the PCR program and remove a 10 &#956;l aliquot at pre-determined cycle numbers to monitor for appearance of desired product. Typically, cycles 10, 13, and 16 are chosen for testing from a total RNA sample.</li>
	</ol>
	</li>
	<li>Prepare an 8% non-denaturing acrylamide gel by mixing 2 ml of 40% acrylamide, 1 ml of 10x&#160;TBE, and H2O to 10 ml. Then add 100 &#956;l of 10% APS and 10 &#956;l of TEMED. Pour gel and let set.
	<ol>
		<li>Pre-run the gel at 12 V/cm of gel width for 30 min. Flush wells. Add 1 &#956;l of 10x&#160;loading buffer (15% Ficoll-400, 66 mM EDTA, 20 mM Tris-HCl pH 8.0, 0.1% (w/v) SDS, 0.01% (w/v) bromophenol blue), add 10 &#956;l of sample to each well of dimensions 5 mm x 1 mm x 15 mm. Typically, each well can load up to 30 &#956;l of samples. Run the sample gel at 10 V/cm of gel width in 1x&#160;TBE buffer until the bromophenol blue dye just runs out of the bottom of the gel.</li>
		<li>Stain and visualize gel as described in step 2.3.</li>
		<li>Excise the 146 base pair band and elute overnight in 0.25 ml of HSCB in a fashion similar to what is described in section 2.4. Precipitate DNA in an equal volume of isopropanol and 1/10th volume sodium acetate. Wash pellet in 70% ethanol and resuspend in TE buffer.</li>
		<li>Quantify the library using picogreen according to manufacturer&#8217;s recommendations.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Hafner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-ligase-dependent+biases+in+miRNA+representation+in+deep-sequenced+small+RNA+cDNA+libraries.">RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries.</a> RNA. 17 (9), 1697-1712 (2011).</li><li>Zhang, Z., Lee, J. E., Riemondy, K., Anderson, E. M., Yi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+RNA+cloning+enables+accurate+quantification+of+miRNA+expression+by+deep+sequencing.">High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing.</a> Genome Biology. 14 (10), 109 (2013).</li><li>Consortium, T. E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ENCODE+(ENCyclopedia+Of+DNA+Elements)+Project.">The ENCODE (ENCyclopedia Of DNA Elements) Project.</a> Science. 306 (5696), 636-640 (2004).</li><li>Consortium, T. E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2012).</li><li>Linsen, S. E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+and+possibilities+of+small+RNA+digital+gene+expression+profiling.">Limitations and possibilities of small RNA digital gene expression profiling.</a> Nature Methods. 6 (7), 474-476 (2009).</li><li>Jayaprakash, A. D., Jabado, O., Brown, B. D., Sachidanandam, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+remediation+of+biases+in+the+activity+of+RNA+ligases+in+small-RNA+deep+sequencing.">Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing.</a> Nucleic Acids Research. 39 (21), 141 (2011).</li><li>McLaughlin, L. W., Romaniuk, E., Romaniuk, P. J., Neilson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Acceptor+Oligoribonucleotide+Sequence+on+the+T4+RNA+Ligase+Reaction.">The Effect of Acceptor Oligoribonucleotide Sequence on the T4 RNA Ligase Reaction.</a> European Journal of Biochemistry. 125 (3), 639-643 (1982).</li><li>Zhuang, F., Fuchs, R. T., Sun, Z., Zheng, Y., Robb, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+bias+in+T4+RNA+ligase-mediated+3&#8242;-adapter+ligation.">Structural bias in T4 RNA ligase-mediated 3&#8242;-adapter ligation.</a> Nucleic Acids Research. 40 (7), 54 (2012).</li><li>Harrison, B., Zimmerman, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer-stimulated+ligation:+enhanced+ligation+of+oligo-+and+polynucleotides+by+T4+RNA+ligase+in+polymer+solutions.">Polymer-stimulated ligation: enhanced ligation of oligo- and polynucleotides by T4 RNA ligase in polymer solutions.</a> Nucleic Acids Research. 12 (21), 8235-8251 (1984).</li><li>Torchia, C., Takagi, Y., Ho, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Archaeal+RNA+ligase+is+a+homodimeric+protein+that+catalyzes+intramolecular+ligation+of+single-stranded+RNA+and+DNA.">Archaeal RNA ligase is a homodimeric protein that catalyzes intramolecular ligation of single-stranded RNA and DNA.</a> Nucleic Acids Research. 36 (19), 6218-6227 (2008).</li><li>Lau, N. C., Lim, L. P., Weinstein, E. G., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Abundant+Class+of+Tiny+RNAs+with+Probable+Regulatory+Roles+in+Caenorhabditis+elegans.">An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans.</a> Science. 294 (5543), 858-862 (2001).</li><li>Yi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DGCR8-dependent+microRNA+biogenesis+is+essential+for+skin+development.">DGCR8-dependent microRNA biogenesis is essential for skin development.</a> Proceedings of the National Academy of Sciences. 106 (2), 498-502 (2009).</li><li>Leung, A. K. 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The authors declare no competing financial interests.

The methodology described herein makes use of several key variables to maximize ligation efficiencies, namely high concentrations of PEG, use of randomized linkers, and high concentration of linkers2,6,9. This approach permits reliably quantitative sequencing libraries from total RNA samples2. We have conducted multiple titrations of input RNA and have concluded that the preceding methodology is best suited for total RNA amounts in the 1-8 &#956;g range (data not shown). When amounts in the 10-500 n...

High-throughput sequencing based methodologies have been widely applied to many biological samples in recent years greatly expanding our understanding of the molecular complexity of biological systems3,4. However, preparation of RNA samples for high-throughput sequencing often imparts specific biases inherent to the employed methodology, limiting the potential utility of these powerful techniques. These method specific biases have been well documented for ligation-based, small-RNA library preparations1,2,5,6. These biases result in 1,000-fold variation in reads numbers for equimolar synthetic miRNAs, making inference of miRNA abundance from sequencing data wildly variable and error prone.
Studies focusing on the properties of phage-derived T4 RNA ligases have documented that the enzymes exhibit nucleotide-based preferences7, which manifest as biased libraries in high-throughput sequencing experiments1,2,8. In order to minimize the biases imparted by RNA ligases, multiple strategies have been employed; macromolecular crowding9, randomizing the nucleotide sequence on the adapter which is proximal to the ligation site6, and employing high concentrations of ligation adapter2. Through a combination of these three approaches we have developed a work-flow for unbiased preparation of small RNA libraries compatible for high-throughput sequencing (Figure 1). For direct comparisons between current protocols and our optimized method, please refer to our recent report2. This optimized method yields ligation efficiencies of greater than 95% at both 3&#8217; and 5&#8217; steps and permits the unbiased ligation of small RNA molecules from synthetic and biological samples2.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name of Reagent/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:280px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">3&#39; Linker (5&#39; phosphorylated, 3&#39; blocked)</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">5&#39; Linker</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>5&#39; blocked, HPLC Purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">T4RNL2 (1-249 K227Q)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0351S</td>
			<td>Specialized for ligation of pre-adenylated DNA adapters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">10X Ligation Buffer (without ATP)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0351S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">10X Ligation Buffer (with ATP)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">RNaseOUT&#160;</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">10777-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Polyethylene Glycol (mol. Wt. 8000)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Nuclease-free water</td>
			<td style="width:175px;">Ambion</td>
			<td style="width:157px;">AM9937</td>
			<td>We have found water collected from a distillation apparatus to be of equvalent quality.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">T4RNL1</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Superscript III RT kit</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">18080-051&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Phusion PCR kit</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0530S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina RP1 Primer</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina RT Primer</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina Index Primer(s)</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">40% Acrylamide</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:157px;">BP14081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Urea</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">U6504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Ammonium persulfate</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tetramethyethylenediamine (TEMED)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">2X Denaturing RNA loading buffer</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0351S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Razor blades</td>
			<td style="width:175px;">VWR</td>
			<td style="width:157px;">55411-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">SpinX Centricon Tubes</td>
			<td style="width:175px;">Costar</td>
			<td style="width:157px;">CLS8161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Low Retention Microfuge tubes</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:157px;">02-681-320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sybr Gold</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">S-11494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Adenylation Kit</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">E2610L</td>
			<td></td>
		</tr>
	</tbody>
</table>

highly efficient ligation of small rna molecules for microrna quantitation by high-throughput sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

The anticipated results for the preceding method should initially be observation of a single nucleotide shift (increase) in the size of the DNA oligonucleotide that was subject to adenylation by Mth RNA ligase (Figure 2). Following 3&#8217; ligation, visualization of the acrylamide gel indicates (see Figure 3) sharp high molecular weight bands evident in the 100-300 nucleotide region of the gel. This indicates that the total RNA sample being used is of high quality (not degraded...

Watch this Scientific Journal Video about Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing at JoVE.com

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

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11.8K Views.  University of Colorado, Boulder. MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs. 

Video: Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...