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[DESCRIPTION]
The NEXTflex&trade; Rapid Directional qRNA-Seq Kit is designed to prepare directional, strandspecific RNA libraries for sequencing using Illumina&reg; sequencers while enabling the high precision measurement of RNA molecules.&nbsp;<br /><br />Please see the <a href="http://www.biooscientific.com/Portals/0/Manuals/NGS/5130-01D-NEXTflex-Rapid-Directional-qRNA-Seq-Kit.pdf" target="_blank">full manual</a> for additional details.

[GUIDELINES]
<strong>OVERVIEW:</strong><br />The NEXTflex&trade; Rapid Directional qRNA-Seq Kit is designed to prepare directional, strandspecific RNA libraries for sequencing using Illumina&reg; sequencers while enabling the high precision measurement of RNA molecules. The NEXTflex&trade; Rapid Directional qRNA-Seq Kit efficiently generates libraries equivalent to conventional directional, strand-specific RNASeq libraries, but with the added feature of Molecular Indexing&trade;, a DNA labeling technology developed by Cellular Research, Inc. Directionality is retained by adding dUTP during the second strand synthesis step and subsequent cleavage of the uridine-containing strand using Uracil DNA Glycosylase. The strand that&rsquo;s sequenced is the cDNA strand. This kit contains a set of 96 distinct molecular labels on the sequencing adapters. Each label consists of an 8 nucleotide barcode tag. During the ligation reaction, each cDNA fragment end independently and randomly ligates to a single label from this pool of 96 adapters to result in a total of 96 x 96 = 9,216 possible combinations across both ends. For every clone sequenced, paired-end reads reveal the chosen label on each end along with adjoining cDNA sequence. This allows for differentiation between re-sampling of the same molecule and sampling of a different molecule of identical sequence. Analysis using molecular indexing information provides an absolute, digital measurement of gene expression levels, irrespective of common amplification distortions observed in many RNA-Seq experiments. In addition to encoding cDNA fragments at the molecular level, the kit also allows for the application of sample-specific barcodes during the library preparation PCR step. A more detailed description of the use of molecular indexing for RNA-Seq is available in our product application note, Directional qRNA-Seq: Combining the Power of Stranded RNA-Seq with the Quantitative Precision of Molecular Labels, available for download here or contact us at BiooNGS@BiooScientific.com. <br /><br />For deconvolution of unique read fragments, Bioo Scientific now offers a dqRNASeq script. Using read pairs aligned to transcripts and FASTQ files, this script will generate a table listing fragments, start/stop sites (USS) in transcripts, and molecular labels (STL). The script will also generate a table listing the total number of read pairs per transcript and the number of read pairs after STL, USS, and STL/USS correction. The script can be downloaded <a href="BiooScientific.com" target="_blank">here</a> or contact us at BiooNGS@BiooScientific.com.<br /><strong><br /><br />Required Materials not Provided <br /></strong>&bull;Total RNA Input: 10 ng &ndash; 1 &micro;g total RNA for enrichment by NEXTflex&trade; Poly(A) Beads (Cat. # 512979, 512980, 512981). NEXTflex&trade; Poly(A) Beads are available separately. <br />&bull; mRNA Input: ~1 ng - 100 ng isolated mRNA. <br />&bull; rRNA-depleted RNA Input: ~1ng - 100 ng. Bioo Scientific recommends Ribo-Zero&trade; (Epicentre) or RiboMinus&trade; (Life Technologies) for rRNA depletion. <br />&bull; 100% Ethanol (stored at room temperature) <br />&bull; 80% Ethanol (stored at room temperature) <br />&bull; 2, 10, 20, 200 and 1000 &micro;L pipettes <br />&bull; RNase-free pipette tips <br />&bull; Nuclease-free 1.5 mL microcentrifuge tubes <br />&bull; Thin wall nuclease-free 0.5 mL microcentrifuge tubes <br />&bull; 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar <br />&bull; Adhesive PCR Plate Seal (BioRad, Cat # MSB1001) <br />&bull; Agencourt AMPure XP 60 mL (Beckman Coulter Genomics, Cat # A63881) <br />&bull; Magnetic Stand -96 (Ambion, Cat # AM10027) or similar for post PCR cleanup <br />&bull; Microcentrifuge <br />&bull; Thermocycler <br />&bull; Heat block <br />&bull; Vortex<strong><br /><br />Revision History</strong><br />
<table>
<tbody>
<tr>
<td>Version</td>
<td>Date</td>
<td>Description</td>
</tr>
<tr>
<td>V14.03</td>
<td>March 2014</td>
<td>Initial Product Launch</td>
</tr>
<tr>
<td>V14.07</td>
<td>July 2014</td>
<td>The Molecular Indices/Adapters, post-adapter ligation cleanup and PCR parameters have been optimized to improve efficiency. Please ensure PCR programs are updated.</td>
</tr>
<tr>
<td>V14.09</td>
<td>September 2014</td>
<td>The starting material necessary for RNA has been reduced to 10 ng total RNA when poly(A) enriched over NEXTflex&trade; Poly(A) Beads (Cat. # 512979, 512980, 512981).</td>
</tr>
<tr>
<td>V15.04</td>
<td>April 2014</td>
<td>The name of the NEXTflex&trade; Directional qRNA Molecular Labels (1 &micro;M) has been changed to the NEXTflex&trade; Molecular Index Adapters (1 &micro;M).</td>
</tr>
</tbody>
</table>
<br /><br /><strong>Contents, Storage and Shelf Life</strong> <br />The NEXTflex&trade; Rapid Directional qRNA-Seq&trade; Kit contains enough material to prepare 8 RNA samples for Illumina&reg; compatible sequencing. The shelf life of all reagents is 12 months when stored properly. All components can safely be stored at -20&deg;C.&nbsp;<br /><br /><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/wjc3kn.png" alt="" /><br /><br /><strong>Required Materials not Provided</strong><br /> &bull; Total RNA Input: 10 ng &ndash; 1 &micro;g total RNA for enrichment by NEXTflex&trade; Poly(A) Beads (Cat. # 512979, 512980, 512981). NEXTflex&trade; Poly(A) Beads are available separately. <br />&bull; mRNA Input: ~1 ng - 100 ng isolated mRNA. <br />&bull; rRNA-depleted RNA Input: ~1ng - 100 ng. Bioo Scientific recommends Ribo-Zero&trade; (Epicentre) or RiboMinus&trade; (Life Technologies) for rRNA depletion. <br />&bull; 100% Ethanol (stored at room temperature) <br />&bull; 80% Ethanol (stored at room temperature) <br />&bull; 2, 10, 20, 200 and 1000 &micro;L pipettes <br />&bull; RNase-free pipette tips <br />&bull; Nuclease-free 1.5 mL microcentrifuge tubes <br />&bull; Thin wall nuclease-free 0.5 mL microcentrifuge tubes <br />&bull; 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar <br />&bull; Adhesive PCR Plate Seal (BioRad, Cat # MSB1001) <br />&bull; Agencourt AMPure XP 60 mL (Beckman Coulter Genomics, Cat # A63881) <br />&bull; Magnetic Stand -96 (Ambion, Cat # AM10027) or similar for post PCR cleanup <br />&bull; Microcentrifuge <br />&bull; Thermocycler <br />&bull; Heat block <br />&bull; Vortex<br /><br /><strong>NEXTflex&trade; Rapid Directional qRNA-Seq&trade; Sample Preparation Flow Chart</strong><br /><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/wkc3kn.png" alt="" /><br /><br /><strong>Starting Material</strong> <br />The NEXTflex&trade; Rapid Directional qRNA-Seq&trade; Kit has been optimized and validated using poly(A) enriched or rRNA depleted RNA (~1 ng - 100 ng). Only 10 ng - 1 &micro;g of total RNA are required if NEXTflex&trade; Poly(A) beads are used to enrich for mRNA*. Bioo Scientific recommends examining total RNA integrity using an Agilent Bioanalyzer. High quality total RNA preparations should have an RNA Integrity Number (RIN) greater than or equal to 8. Alternatively, total RNA may be run on a 1 - 2% agarose gel and integrity examined by staining with ethidium bromide. High quality RNA should have a 28S band that is twice as intense as the 18S band of ribosomal RNA. Lower amounts of starting material result in higher duplication rates and other changes in sequencing data quality.<br /> *Low RNA inputs may reduce library complexity. Read our application note about constructing high quality RNA-Seq libraries from limited amounts of total RNA. Request a PDF copy by emailing BiooNGS@BiooScientific.com.<br /><br /><strong>Library Validation<br /><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/w5c3kn.png" alt="" /><br /></strong>

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