SEQuoia Complete Stranded RNA Library Prep Kit

The SEQuoia Complete RNA Library Prep Kit provides all reagents required to construct a library from RNA, with the exception of the SPRISelect beads from Beckman Coulter.

The following reagents are required but not supplied. These materials have been validated with the SEQuoia Complete Stranded RNA Library Prep Kit protocol. Substitutions may not produce ideal results.

• 2 ml RNase, DNase free, low-bind PCR tube strips and cap strips (TBC0802 or TBC1202)
• RNase-free filtered pipet tips: 10 μl, 20 μl, and 200 μl
• SPRISelect beads from Beckman Coulter
• 10mM Tris-HCl pH8 (RNase, DNase Free)
• 80% ethanol (prepare fresh)

Optional Materials Not Provided

• SEQuoia Dual Indexed Primers Set (12011928) or SEQuoia Dual Indexed Primers Plate (12011930)
• rRNA depletion kit
• ddPCR Library Quantification Kit for Illumina TruSeq (1863040)

Equipment List

• Thermal cycler for accurate incubation temperatures
• Calibrated single-channel and multi-channel pipets: 10 μl, 20 μl, and 200 μl
• Magnetic rack for small-scale separation of magnetic beads in 0.2 ml tubes
• Vortex
• Microcentrifuge for 0.2 ml tubes
• 96-well PCR cooler rack
• Ice bucket

The ideal RNA purification strategy depends on the sample type and experimental objectives. We have tested and recommend using PureZOL RNA Isolation Reagent (7326880) for tissue samples and the SingleShot Cell Lysis Kit (1725080) for cultured cells. If small RNAs are of interest, we do not recommend using a column-based based kit, as RNA fragments under 200 may be lost. Instead, use the SingleShot Cell Lysis Kit.

Additionally, these references provide a good overview of various methods for small RNA isolation;

• Quantification of Small Non-Coding RNAs Allows an Accurate Comparison of miRNA Expression Profiles. Masotti A, et al. Journal of Biomedicine and Biotechnology Volume 2009, Article ID 659028
• A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples. Sellin Jeffries MK, Kiss AJ, Smith AW, Oris JT. BMC Biotechnology, 201414:94

The SEQuoia Complete Stranded RNA Library Prep kit is compatible with 1 ng to 1 μg of total RNA (pre-depleted of ribosomal RNA)

Typically, SEQuoia Complete Stranded RNA Library Prep Kit requires 3–5 fewer cycles compared to other commercially available kits, due to a high conversion rate. This is advantageous because over amplification can result in artifacts such as amplification bias, increased duplicate rates, and chimeras. To minimize artifacts, optimize the number of PCR amplification cycles so that the final library concentration is between 200–1,000 ng. The number of PCR cycles is dependent upon the amount and quality of input RNA. The following table provides general guidelines and can be used as a starting point for optimization.

PCR Cycle Recommendations

SEQuoia Complete Stranded RNA Library Prep Kit captures both short and long RNA biotypes, including mRNA, miRNA, lncRNA, snoRNA. Because this kit efficiently captures small RNAs, it is recommended that sequencing depth be 20M–30M reads to ensure the accuracy of the results.

We recommend sequencing with single-end reads to save on sequencing cost.

• The first 8 bases on R2 are a random tag sequence which can be used as a UMI.
• Recommended read length: 100 x 8 or 75 x 8 of Read1 x Read2
• Read depth should be determined based on the application and prior experience. The typical range is 20–30M single-end reads due to the number of small RNA fragments captured, which increases the library complexity.
• With single reads, using SEQuoia Complete Stranded RNA Library Prep Kit, you can get coverage of full transcript as shown with all the data on this page.
• For pair-end reads, there may be a significant drop in read quality for R2 because of the presence of the poly(T) sequence.

No. The adapters in the SEQuoia Complete Stranded RNA Library Prep Kits are compatible with flow cells used with Illumina platforms.