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Guidelines for Small RNA Sequencing Projects

The High Throughput Genomics Core Facility supports two options for small RNA-focused library preparation using kits by New England BioLabs and by Illumina. All sample preparation kits supported by the core facility include indexed adapters which enable sequencing of multiple libraries in the same lane. A typical Illumina sequencing lane using version 4 chemistry delivers approximately 230-270 million reads. For small RNA profiling projects, we recommend multiplexing libraries such that each individual library is represented by approximately 20 million reads. On average 50-60% of the sequence reads from small RNA sequencing projects will align to the genome when the data is filtered for reads that are 15 bases or longer. Guidelines for small RNA sequencing projects are described below:
  1. Small RNA Sequencing Library Prep Kits supported by the High Throughput Genomics Core Facility are described below:
    1. The NEBNext Multiplex Library Prep Set for Illumina recommends an input of 100 to 1000 ng of total RNA in a volume of 6 ul for library construction (please provide a total volume of 10 ul to enable quality control assays on your sample). Following pcr amplification of adapter-ligated cDNA, the library is size selected on a Sage Science Pippin Prep cassette to enable enrichment of inserts that represent miRNA and piRNA. Purification by the Pippin Prep enables a higher level of consistency from sample to sample compared to conventional methods for this enrichment step. This kit works best with total RNA samples containing a RIN number of 6 or higher.
    2. The Illumina TruSeq Small RNA Sample Preparation Kit is optimized for an input of 1000 ng of total RNA. Following pcr amplification of the adapter-ligated cDNA, the library is resolved on a polyacrylamide gel and the fraction representing miRNA and piRNA is excised and eluted from the gel. This kit works best with total RNA samples containing a RIN number of 6 or higher.
  2. Purification of RNA: Total RNA should be purified using a column-based kit such as the Qiagen miRNeasy Mini Prep kit, the Zymo Research Direct-zol MiniPrep Kit, or the Ambion mirVana miRNA Isolation kit. In all cases. it is recommended to include a DNase treatment step to avoid background reads contributed by contaminating DNA.
  3. Avoid Organic Extraction Methods: The use of organic extraction reagents, such as Trizol, as a stand-alone method for RNA purification is discouraged. The quality of RNA purified by these methods tend to be lower due to co-precipitation of contaminants during the EtOH precipitation step. Co-precipitated contaminants may result in an elevated A260 value on a NanoDrop spectrophotometer due to the absorbance contributions from the contaminating molecules. Furthermore, organic contaminants that co-purify with the RNA can have an inhibitory step on enzymatic steps included in small RNA library construction and library prep of these samples may occasionally fail.
  4. Assessment of RNA Quality: Prior to constructing an Illumina sequencing library, total RNA quality is validated by running an aliquot an on Agilent 2200 TapeStation. It is recommended that RNA samples have a RIN number of 6.0 or higher if they are to be used for small RNA library preparation. Samples with lower RIN number will have an increasingly higher percent of degraded RNA products in the size range of small RNA species (15-30 nucleotides). Being of similar size to miRNA and piRNA species, these contaminating products derived from RNA degradation may present a substantial contribution to the size selected small RNA library. The client will be contacted after the TapeStation run and prior to library preparation if there are any concerns about the quality of the RNA for the library prep method that was selected.
  5. Library QC: Following construction of a Small RNA sequencing library, the library is validated by running an aliquot on an Agilent 2200 TapeStation and by defining the molarity of the library using a qPCR assay (Kapa Biosystems Library Quantification Kit for Illumina). After normalizing library concentrations following the completion of these two assays, libraries can be pooled in preparation for sequencing.