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[DESCRIPTION]
This protocol is for the NEXTflex™ Small RNA Sequencing Kit v2, designed to prepare small RNA libraries for sequencing using Illumina® sequencers. This kit utilizes adapters with randomized ends to greatly reduce sequence bias in small RNA sequencing library construction.<br /><br />Please see the <a href="http://www.biooscientific.com/Portals/0/Manuals/NGS/5132-03-NEXTflex-Small-RNA-Seq-Kit-v2.pdf" target="_blank">full manual</a> for additional details.<br /><br />(The protocol below describes preparation of small RNA sequencing libraries from <strong>Total RNA</strong> starting material; for  small RNA samples, please see <a href="https://www.protocols.io/view/NEXTflex-Small-RNA-Sequencing-for-Small-RNA-Starti-dre53d" target="_blank">this</a> protocol.)

[GUIDELINES]
<strong>Contents, Storage and Shelf Life </strong><br />The NEXTflexÃ¢ÂÂ¢ Small RNA Sequencing Kit v2 contains enough material to prepare 24 RNA samples for IlluminaÃÂ® compatible next-generation sequencing. The shelf life of all reagents is 6 months when stored properly. All components can safely be stored at -20ÃÂ°C, except: Adapter Depletion Solution, Resuspension Buffer, Nuclease-free Water, and Elution Buffer, which can be stored at room temperature.<br /><br /><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/w7c3kn.png" alt="" width="459" height="255" /><br /><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/w9c3kn.png" alt="" /><br />*see Reagent Preparation for dilution procedure<br /><br /><strong>Required Materials Not Provided</strong> <br />Ã¢ÂÂ¢ 1-10 ÃÂµg total RNA or purified small RNA from 1-10 ÃÂµg total RNA in up to 4 ÃÂµL Nucleasefree Water <br />Ã¢ÂÂ¢ Isopropanol <br />Ã¢ÂÂ¢ 80% Ethanol <br />Ã¢ÂÂ¢ 2, 10, 20, 200 and 1000 ÃÂµL pipettes <br />Ã¢ÂÂ¢ RNase-free pipette tips <br />Ã¢ÂÂ¢ Microcentrifuge <br />Ã¢ÂÂ¢Nuclease-free 1.7 mL microcentrifuge tubes <br />Ã¢ÂÂ¢ 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar <br />Ã¢ÂÂ¢ Sterile disposable pestles (Fisher Cat # K749521-1500 or similar) <br />Ã¢ÂÂ¢ Thin wall nuclease-free PCR tubes <br />Ã¢ÂÂ¢ Thermocycler <br />Ã¢ÂÂ¢ Heat block <br />Ã¢ÂÂ¢ 6% TBE PAGE gels (1.0 mm) (Life Technologies Cat # EC6265BOX) <br />Ã¢ÂÂ¢ 1X TBE buffer<br />Ã¢ÂÂ¢ Clean razor or scalpel <br />Ã¢ÂÂ¢ Nucleic acid stain such as SYBR Gold (Invitrogen) <br />Ã¢ÂÂ¢ UV transilluminator or gel documentation instrument <br />Ã¢ÂÂ¢ Gel Electrophoresis apparatus <br />Ã¢ÂÂ¢ Electrophoresis power supply <br />Ã¢ÂÂ¢ Vortex<br /> Ã¢ÂÂ¢ 0.45ÃÂµm, 2 mL Spin-X Centrifuge tube (Sigma Cat # CLS8162) <br />Ã¢ÂÂ¢ Agencourt AMPure XP 60 mL (Beckman Coulter Genomics, Cat # A63881) <br />Ã¢ÂÂ¢ Magnetic Stand -96 (Ambion, Cat # AM10027) or similar <br />Ã¢ÂÂ¢ Magnetic stand for micrcentrifuge tubes (Life Technologies DynaMagÃ¢ÂÂ¢-2 or similar)<br /><br /><strong>Optional Materials Not Provided </strong><br />Ã¢ÂÂ¢ÃÂ If multiplexing: NEXTflexÃ¢ÂÂ¢ Small RNA Barcodes A Ã¢ÂÂ D (513305, 513306, 513307, 513308)<br /><br /><strong>NEXTflexÃ¢ÂÂ¢ Small RNA Sample Preparation Flow Chart<br /><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/xbc3kn.png" alt="" width="364" height="569" /><br /></strong><br /><strong>Starting Material</strong> <br />The NEXTflexÃ¢ÂÂ¢ Small RNA Sequencing Kit v2 has been optimized and validated using total RNA (1 - 10 ÃÂµg), purified small RNA (from 1 - 10 ÃÂµg total RNA), and a synthetic miRNA pool (Ã¢ÂÂ¥100 pg). Best results are obtained with high quality starting material. The use of degraded RNA may result in poor yields or lack of sequencing output data. Bioo Scientific recommends running total RNA on a 1 - 2% agarose gel or examining its integrity using an Agilent Bioanalyzer. High quality total RNA preparations should have an 28S band that is twice as intense as the 18S band of ribosomal RNA. At low concentrations, small RNA is difficult to detect on a gel; however it can be detected using an Agilent Bioanalyzer Small RNA assay (see Figure 2 in Appendix A). We recommend beginning with total RNA or a preparation of highly enriched small RNA by PAGE selecting small RNAs around 15-45 nt long or using a phenol-based small RNA isolation method such as BiooPureÃ¢ÂÂ¢ (Cat # 5301) for enrichment. <br /><br />If the user is performing the procedure for the first time, we recommend using the microRNA Control included in the kit: 5Ã¢ÂÂPhos/CUCAGGAUGGCGGAGCGGUCU/3Ã¢ÂÂ. This positive control sample consists of 21 RNA nucleotides and does not match any known sequence in miRBase. When running a positive control reaction, the user should add 1 ÃÂµL of the microRNA Control in STEP A instead of their small RNA sample and expect to observe a 147 nt PCR product. The microRNA control may degrade with multiple freeze thaw cycles or exposure to nucleases. If you plan on using the control multiple times, we recommend aliquoting into several tubes and storing at -20ÃÂ°C. For a total RNA positive control, human brain total RNA (Ambion catalog number AM7962 or similar) is recommended. <br /><br /><strong>Reagent Preparation</strong> <br />1. Vortex and micro centrifuge each component prior to use, to ensure material has not lodged in the cap or the side of the tube. <br />2. Add 18 mL of molecular biology-grade water to each bottle of Elution Buffer (10X) to make a 1X Elution Buffer. Check box on bottle to show water has been added.<strong> <br /><br />Small RNA Starting Material <br /></strong>1. Add 10 ÃÂµL of Nuclease-free Water to each sample and mix well by pipette.<br /> 2. Add 6 ÃÂµL of Adapter Depletion Solution and mix well by pipette. <br />3. Add 40 ÃÂµL of AMPure XP beads and 60 ÃÂµL of ispropanol and mix well by pipette. <br />4. Incubate for 5 minutes. <br />5. Magnetize sample until solution appears clear. <br />6. Remove and discard supernatant.<br />7. Add 180 ÃÂµL of freshly prepared 80% ethanol, incubate for 30 seconds, and remove with a P200 or P300 set to 200 ÃÂµL. Repeat this step for a total of 2 ethanol washes. IMPORTANT: Always use freshly prepared 80% ethanol and do not incubate the bead pellet with 80% ethanol for extended periods. <br />8. Incubate sample for 3 minutes. After one minute, remove all residual liquid that may have collected at the bottom of the well. <br />9. Remove plate from magnetic stand and resuspend bead pellet in 22 ÃÂµL of Resuspenion Buffer by pipetting volume up and down. Ensure that beads are completely resuspended. <br />10. Incubate 2 minutes. During incubation, add 6 ÃÂµL of Adapter Depletion Solution to a new, empty well. <br />11. Magnetize sample until solution appears clear. <br />12. Transfer 20 ÃÂµL of supernatant to a new well containing 6 ÃÂµL Adapter Depletion Solution and mix well by pipette. <br />13. Add 40 ÃÂµL of AMPure XP beads and 60 ÃÂµL of ispropanol and mix well by pipette. <br />14. Incubate for 5 minutes. <br />15. Magnetize sample until solution appears clear. <br />16. Remove and discard supernatant. <br />17. Add 180 ÃÂµL of freshly prepared 80% ethanol, incubate for 30 seconds, and remove with a P200 or P300 set to 200 ÃÂµL. Repeat this step for a total of 2 ethanol washes. IMPORTANT: Always use freshly prepared 80% ethanol and do not incubate the bead pellet with 80% ethanol for extended periods. <br />18. Incubate sample for 3 minutes. After one minute, remove any residual liquid that may have collected at the bottom of the well. <br />19. Remove plate from magnetic stand and resuspend bead pellet in 12 ÃÂµL of Nuclease-free Water by pipetting. Ensure that beads are completely resuspended. <br />20. Incubate for 2 minutes. <br />21. Magnetize until solution appears clear. <br />22. Transfer 11 ÃÂµL of supernatant to a new well. Proceed to Step C: 5Ã¢ÂÂ NEXTflexÃ¢ÂÂ¢ 4N Adapter Ligation. <br /><br /><strong>Figure 2</strong><br />6% TBE-PAGE gel <br />A. PCR product from library constructed from 1 ÃÂµg human brain total RNA <br />B. Ready to Load Low MW Ladder <br />C. PCR product from library constructed from 1 ÃÂµL miRNA control <br />NOTE: 25 bp band not shown. 225 bp &amp; 250 bp bands may run as a single band.<br /><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/xxc3kn.png" alt="" /><br /><strong>References<br /></strong>Jayaprakash et al. <a href="http://nar.oxfordjournals.org/content/early/2011/09/02/nar.gkr693.full" target="_blank">Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing</a>. Nucl. Acids Res. (2011) 39 (21):e141<br /><br /><br /><strong><br /></strong><br /><br /><br />

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