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[DESCRIPTION]
<p><strong>GENERAL INFORMATION</strong></p>
<p> </p>
<p><em><strong>Product Overview</strong></em></p>
<p> </p>
<p>The NEXTflex™ Small RNA-Seq Kit v3 can be used to prepare small RNA libraries from total RNA for Illumina-compatible next-generation sequencing. The NEXTflex Small RNA-Seq Kit v3 is designed to greatly reduce formation of adapter-dimer product in small RNA-seq library preparation, allowing completely gel-free library preparation from typical input amounts, or allowing libraries to be created from low input amounts with a PAGE-based size selection of the final library. This kit utilizes patent-pending adapters with randomized ends to greatly reduce sequence bias in small RNA sequencing library construction (1), allowing more accurate identification and quantification of microRNAs, piRNAs, and other small RNAs.</p>
<p> </p>
<p>This manual includes protocols for <strong>size selection using a gel-free (Step H1)</strong> or <strong>PAGE-based (Step H2)</strong> method. <em><span style="text-decoration: underline;">The gel-free protocol described in this manual is recommended for library preparations where a sufficient amount of ~150 bp product and no ~130 bp adapter-dimer product is seen after 16 cycles of PCR</span></em>, which is typically achieved when using 1μg of total RNA starting material.</p>
<p> </p>
<p><em>Note</em>:</p>
<p>1) This protocol can be adapted for compatibility with sequencing platforms other than Illumina.</p>
<p>The sequences of the adapters (provided in Figure 1) and PCR primers must be modified for the specific sequencing platform.</p>
<p> </p>
<p>2) This protocol is specific for <span style="text-decoration: underline;"><strong>v</strong><strong>ersion 3</strong></span> of the NEXTflex™ Small RNA-Seq Kit:</p>
<p>Bioo Scientific, Catalog # 5132-05 (8 reactions), or Catalog # 5132-06 (48 reactions).</p>
<p>Modifications to the protocol may be required for updated versions of the kit.</p>
<p> </p>
<p>References<br />1. Jayaprakash, A.D., et al., Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Res, 2011. 39(21): p. e141.</p>

[BEFORE_START]
<p>Determine the concentration and quality of the total RNA sample on an Agilent Bioanalyzer, using Agilent RNA nano or pico kits.</p>

[GUIDELINES]
<p>1) Start with 1ug of high quality (determined by Bioanalyzer) total RNA.</p>
<p> </p>
<p>2) All steps with beads should be done carefully, pipetting up and down until the sample and beads are clearly mixed and the beads are evenly distributed throughout the sample.</p>
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<p>3) Ensure that during bead cleanup steps, the ethanol washes are completely removed from the beads.</p>
<p> </p>
<p>4) The NEXTflex Small RNA Sequencing Kit v3 protocol requires 1.5-2 days for completion. Approximate times to complete each step and safe Stopping Points are noted in the protocol; however, careful planning and time management are important for efficient and successful small RNA library preparation. If performing the protocol for the first time, we highly recommend preparing a library with the included microRNA control.</p>
<p> </p>
<p>5) There are bead cleanup steps in this protocol in which supernatants must be either be carried over to downstream steps, or in which they are to be discarded. First time users are encouraged to retain all supernatants from bead cleanups in order to troubleshoot failed experiments.</p>

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