Small RNA-Seq Library Prep Kit for Illumina
The Small RNA-Seq Library Prep Kit provides a protocol for generating small RNA libraries for Illumina sequencing directly from total RNA or enriched small RNA.
Multiplexing of up to 96 Samples
Multiplexing of up to 96 samples is possible with complimentary i7 indexes provided in the kit. This allows you to pool more samples per sequencing lane and perform cost-efficient experiments on the platforms of different scale – from bench top to high throughput instruments.
Gel-free User-Friendly and Fast Protocol
Lexogen’s Small RNA-Seq kit offers a time saving protocol that can be completed within 5 hours, and requires just about 1 hour of your hands-on time. The final library does not need to go through gel purification. Quick and convenient magnetic bead-based purification can optionally be performed to remove adapters dimers for some demanding RNA inputs.
or exosomal RNA) is first subjected to 3’ Adapter ligation.
Frequently Asked Questions
Please find a list of the most frequently asked questions below. If you cannot find the answer to your question here or want to know more about our products, please contact email@example.com or directly under +43 1 345 1212-41 and +1 (0) 603 498-1666.
In general, we would recommend using 100 ng of total RNA input from cells or tissues. For 100 ng input amounts we recommend using 0.5x dilution of 3’ Adapter, 5’ Adapter, and RT-Primer and 50 µl of EtOH.
Current loading amount recommendations are available for the following instruments:
- MiSeq / HiSeq2500 / HiSeq4000: 15 – 20 pM
- NextSeq 500 / 550: 1.8 – 2.5 pM
Protocol guidelines for bead-based purification of Small RNA-Seq libraries can be found in Appendix G (p.25) of the Small RNA-Seq Library Prep Kit User Guide.
Depending on the starting sample and the application, aiming for a minimum of 4-5 million raw reads per sample would be recommended. For low input RNA amounts or libraries with lower small RNA content, higher numbers of reads per sample may be required.
An example workflow for analysis of miRNAs in small RNA-Seq library sequencing data is provided. This workflow was adapted from: Tam et al., (2015) Optimization of miRNA-seq data preprocessing. Briefings in Bioinformatics 16(6). This paper also provides an additional comparison of programs for read mapping (alignment), normalization methods, and differential expression analysis.
The general workflow involves, trimming of adapters, size filtering of trimmed reads, read mapping:
1. Trimming, adapter-removal, and size filtering (BBDuk, http://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/bbduk-guide/)
1.1. Use bbduk.sh to remove the adapter sequence from the 3’ end of the reads and trim homopolymers.
1.2. Split up the reads into short read and long read fractions.
1.2.1. Short reads read into the adapter sequence AND are shorter than 31 bp and are processed for miRNA alignment.
1.2.2. Long reads are kept separate for alignment to the reference genome.
2. Mapping* (BWA, http://bio-bwa.sourceforge.net/, BBMap, https://sourceforge.net/projects/bbmap/)
2.1. Short read fraction
2.1.1. Filter out reads <= 14 bp -> outputs: “filtered reads: too short” statistic
2.1.2. Map the remaining 15 – 31 bp reads to the ribosomal RNA sequences using BWA. -> outputs: “mapped reads” and “unmapped reads”, mapping statistics
2.1.3. Use the unmapped reads from the BWA alignment and map these against the mirBase annotation (http://www.mirbase.org/ftp.shtml; mature miRNAs, species-specific) -> output “mapped reads”, read count statistics, and miRNA-count table.
2.2. Long read fraction
2.2.1. Map long reads against the reference genome using STAR (https://github.com/alexdobin/STAR) -> output: “mapped reads”, read count statistics, gene_biotype statistics (including rRNA sequences), and gene-read table.
* Alternative programs for read mapping can also be used. A comparison of alignment programs for small RNA-Seq data can be found in Tam et al., 2015.
Additional magnetic bead-based size selection can also be used to remove longer library products after the library prep has been completed.
It is not recommended to use RiboCop for rRNA depletion if you intend to use the RNA for downstream small RNA-Seq library prep. This is because the small RNA fraction can be reduced during the purification steps of the RiboCop protocol.
Due to size differences, libraries prepared with the Lexogen Small RNA-Seq Library Prep Kit (or any other small RNA library prep kit) should not be sequenced together with QuantSeq or SENSE libraries. Please refer to the sequencing guidelines for each library type (library adapter details, loading amounts to use, and use of custom sequencing primers, etc), which are provided in our Library Prep Kit User Guides, and online Frequently Asked Questions (FAQs).