1. What do the ScriptSeq™ Complete Kits contain?
The ScriptSeq Complete Kits provide a complete workflow for RNA-Seq library preparation. They include Ribo-Zero™ components to remove ribosomal RNA (rRNA) from total RNA samples, as well as the ScriptSeq v2 Kit for library prep. The ScriptSeq complete Kits are available in both standard (1-5 µg total RNA) and low-input (100 ng to 1 µg total RNA) formats.
2. How are the ScriptSeq™ v1 and v2 kits different?
The ScriptSeq v2 Kit offers important improvements compared to the original ScriptSeq (v1) kit. These include:
- Improved transcript coverage.
- Significantly reduced G bias.
- Lower RNA input, ~500 pg to 50 ng of rRNA-depleted or poly(A)+ RNA.
- Easier to use—fewer pipetting steps, new MasterMixes for many components.
- Lower price.
3. What are the main differences between ScriptSeq kits and Illumina's first-generation TruSeq™ RNA-Seq kits?
Illumina's Tru-Seq libraries are prepared by a modified adaptor-ligation method, in which a double-stranded cDNA library is prepared from poly(A)-selected or rRNA-depleted RNA. After end repair and addition of a single A residue to each end of the cDNA, adaptors that contain the P5/7 PCR priming sites and Read 1 and 2 sequences are ligated to the ends of the cDNA, producing nondirectional libraries. Illumina's TruSeq RNA-Seq libraries are considered the "gold standard" for RNA-Seq analysis and provide excellent data quality (lower coefficient of variation [CV] in coverage); however, the process can take up to 2 days. In contrast, the ScriptSeq process can be completed in as little as 4 hours and produces directional libraries, but with higher %CV in coverage.
4. Does the ScriptSeq process generate directional libraries?
Yes. ScriptSeq kits make directional libraries such that the sequence generated using the Illumina® Read 1 sequencing primer is that of the sense strand of the original RNA. Using the Illumina Read 1 sequencing primer, the first nucleotide read always corresponds to the 5´ nucleotide of the original fragmented RNA molecule.
5. How does the ScriptSeq method generate directional libraries?
After rRNA depletion (e.g., using Epicentre's Ribo-Zero™ Kits), the RNA is reverse-transcribed using random hexamers that include a 5´ tail containing a unique tagging sequence. After cDNA synthesis, a terminal tagging oligonucleotide (TTO) that has a blocked 3´ end is annealed to the 3´ end of the cDNA. Following extension with a DNA polymerase, a second, unique tagging sequence (complementary to the TTO) is added to the 3´ end of the cDNA. The resulting di-tagged cDNA is amplified by PCR using primers that anneal to the two different tagging sequences on the cDNA.
6. How long does it take to create a ScriptSeq library?
The protocol for the ScriptSeq v2 Kit will take approximately 3.5 to 4 hours to complete, from the chemical fragmentation step/first strand cDNA synthesis to quantification of the library. The time will depend on the number of libraries being prepared.
7. How much RNA do I need to create a ScriptSeq v2 library?
ScriptSeq v2 drastically reduced the amount of poly(A)+ or rRNA-depleted RNA required, to a minimum of 500 pg and a maximum of 50 ng, a 500-fold reduction compared to the original ScriptSeq Kit.
8. Will the ScriptSeq kit allow me to use RNA from any source?
Yes. Virtually any RNA larger than 100 nt can be used to create a ScriptSeq library.
9. Can the ScriptSeq kit be used with RNA from FFPE tissues?
Yes. But the smallest fragment size to use from FFPE tissues should be no smaller than about 75 nt.
10. Can I use the ScriptSeq kit to make RNA-Seq libraries from small RNA, such as miRNA?
The ScriptSeq procedure uses random primed cDNA synthesis, which is very inefficient for small, highly structured RNAs, such as miRNA. We do not recommend using the ScriptSeq kit to prepare small-RNA libraries. Instead, use Epicentre's ScriptMiner Small RNA-Seq Kit.
11. Can I fragment the RNA using a method (for example the Ambion kit) other than described in the kit procedure?
Yes. But the RNA must be cleaned up (fragmentation buffer removed) prior to running the ScriptSeq reaction. If you use a different fragmentation procedure, follow the procedure for FFPE RNA described in Appendix 1 of the protocol.
12. Why do you recommend only 10-15 PCR cycles to enrich the library from the di-tagged single-stranded cDNA?
In general, PCR amplification can generate undesirable duplicate amplicons, and cause other artifacts that create statistical errors in the analysis of the library. This is true with any PCR, and is not limited to the ScriptSeq procedure. The linear amplification range tends to extend only to the 15-17 cycle mark, and thus we recommend using the fewest number of cycles necessary to enrich the library, while minimizing duplicate amplicons and other potential PCR-related errors. Based on the range of amount of input RNA used to generate the library, we have found 10-15 cycles to be optimal for balancing yield with the sequence quality of the library.
13. Can I prepare a library without doing PCR?
No. At least one cycle of PCR must be done. The PCR completes the addition of the adaptor sequences, and adds Index or barcode if desired.
14. Can I use other PCR polymerases for the library enrichment instead of the FailSafe™ PCR Polymerase?
While some researchers prefer to use other polymerases, we recommend FailSafe PCR Polymerase to avoid buffer compatibility issues, and make the product easier to support. We also see better library yields using the FailSafe PCR Polymerase.
15. What is the best method for library purification after the PCR step?
We suggest the use of the AMPure™ XP system (Agencourt/Beckman Coulter) to purify the ScriptSeq v2 kit libraries. The AMPure XP System is best at removing the "primer-dimers" that can occur during PCR. We recommend the MinElute™ PCR Purification system (Qiagen) only for purifying libraries made from FFPE RNA with an average size <200 nt. Libraries purified using the MinElute columns will be contaminated with primer-dimers.
16. Will ScriptSeq libraries work on any Illumina sequencer?
ScriptSeq libraries can be sequenced on any sequencer that uses the Tru-Seq V.2 or V.3 chemistry. However, when running a paired-end library, be certain to use the "PE" flow cell. If running only one single-read library, you can use a single-read flow cell. The sequencing primers to use include the following:
- For Read 1, use the genomic DNA Sequencing Primer in Illumina HP6 solution (lane 11 of the V.2/V.3 reagent set on the cBot).
- For Read 2, use the Multiplexing Read 2 Sequencing primer in solution HP7.
- For the index read, use the Index Sequencing primer in HP8.
The library mass generated will be determined by the number of PCR cycles used to enrich the library. As a general rule, using 50 ng of input rRNA-depleted RNA and 12 PCR cycles will produce 300-400 ng of library.
17. Can ScriptSeq v2 libraries be sequenced using the Illumina MiSeq sequencer?
Yes. The new sequencing mixes HP10, 11, and 12 (Read 1, Read 2 and Index 1, respectively) contain primers that are compatible with ScriptSeq v2 libraries.
18. What is best way to quantify the yield of the ScriptSeq library?
There are several ways to quantify ScriptSeq libraries, but the most accurate method is fluorimetry using a Qubit™ or other fluorimeter and the fluorescent dye Pico Green. The Bioanalyzer has a quantification process built into it, but this is extremely reliant on the amount and pipetting/quantitation accuracy of the standard ladder added to the sample cell. A NanoDrop® or other spectrophotometer is not sensitive enough to accurately quantify the library. ScriptSeq libraries may also be quantified using qPCR, e.g., using the Library Quant Kits from KAPA Biosciences.
19. Why do I get so many rRNA reads when sequencing my ScriptSeq library?
The number of rRNA reads is dependent on the amount of rRNA in the sample. The amount of rRNA in the sample is dependent on the method of rRNA removal or poly(A) enrichment. ScriptSeq libraries prepared from RNA treated with Epicentre's Ribo-Zero kit will yield the fewest rRNA reads, generally less than 2%.
20. Why do I get less than 98% directionality in my libraries with the ScriptSeq kit?
The major cause of loss of directionality is DNA contamination in the RNA preparation. The RNA should be treated with DNase I to remove all traces of DNA, and then the DNase I should be removed, prior to starting the ScriptSeq reaction.