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Guidelines for RNA Sequencing Projects

The High Throughput Genomics Core Facility supports multiple options for gene expression profiling and transcriptome assembly projects through the construction of mRNA-focused libraries from total RNA. 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 gene expression profiling projects, we recommend multiplexing libraries such that each individual library is represented by approximately 20 million reads. Libraries prepared using Ribo-Zero to remove rRNA may require 20-30% more reads. Guidelines for RNA sequencing projects are described below:
  1. RNA Sequencing Library Prep Kits supported by the High Throughput Genomics Core Facility are described below:
    1. The Illumina TruSeq RNA Sample Prep Kit v2 with oligo(dT) selection recommends an input of 100 to 1000 ng of total RNA in a volume of 30 ul for library construction. Libraries constructed with this kit are non-stranded (sequence reads occur in both the sense and anti-sense orientation relative to mRNA) and library preparation begins with the purification of mRNA using oligo(dT) magnetic beads. Following purification, the mRNA is chemically fragmented and random primed for reverse transcription. The average insert size of libraries constructed with the Illumina TruSeq RNA Sample Prep Kit v2 is approximately 150 bp with inserts ranging from 100-400 bp. This kit works best with total RNA samples containing a RNA Integrity Number of 8.0 or higher.
    2. The Illumina TruSeq Stranded mRNA Sample Prep Kit with oligo(dT) selection recommends an input of 100 to 1000 ng of total RNA in a volume of 30 ul for library construction. Libraries constructed with this kit are stranded (sequence reads occur in the same orientation as anti-sense RNA) and library preparation begins with the purification of mRNA using oligo(dT) magnetic beads. Following purification, the mRNA is chemically fragmented and random primed for reverse transcription. The average insert size of libraries constructed with the Illumina TruSeq Stranded mRNA Sample Prep Kit is approximately 150 bp with inserts ranging from 100-400 bp. This kit works best with total RNA samples containing a RNE Integrity Number of 8.0 or higher.
    3. The Illumina TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Gold recommends an input of 100 to 1000 ng of total RNA in a volume of 10 ul for library construction. Libraries constructed with this kit are stranded (sequence reads occur in the same orientation as anti-sense RNA) and library preparation begins with the removal of cytoplasmic and mitochondrial rRNA using Ribo-Zero Gold. Ribo-Zero Gold was designed to remove rRNA molecules from human, mouse and rat samples however, it also works well on RNA samples purified from many other organisms. A species compatibility chart can be found on the Epicentre website (www.epibio.com) many other Following the removal of these RNA species, the remaining RNA is chemically fragmented and random primed for reverse transcription. The average insert size of libraries constructed with the Illumina TruSeq Stranded Total RNA Sample Prep Kit is approximately 150 bp with inserts ranging from 100 to 400 bp. This kit works well with high quality RNA samples as well as degraded RNA samples such as those purified from FFPE tissues. When using this kit, it is essential to DNase treat samples prior to library preparation to minimize the contribution of sequence reads that align to intergenic DNA regions. Although this kit removes rRNA sequences, intronic sequence reads will be present and RNA samples purified from formalin fixed paraffin embedded tissues may routinely yield up to 50% intronic reads.
    4. The Illumina TruSeq Total RNA Sample Prep Kit with Ribo-Zero (Plant) recommends an input of 100 to 1000 ng of total RNA in a volume of 10 ul for library construction. Libraries constructed with this kit are stranded (sequence reads occur in the same orientation as anti-sense RNA) and library preparation begins with the removal of rRNA and mtRNA using RiboZero-Plant. Following the removal of these RNA species, the remaining RNA is chemically fragmented and random primed for reverse transcription. The average insert size of libraries constructed with the Illumina TruSeq Stranded Total RNA Sample Prep Kit is approximately 150 bp with inserts ranging from 100 to 400 bp. This kit works well with high quality RNA samples as well as degraded RNA samples such as those purified from FFPE tissues. When using this kit, it is essential to DNase treat samples prior to library preparation to minimize the contribution of sequence reads that align to intergenic DNA regions. Although this kit removes rRNA sequences, intronic sequence reads will be present and RNA samples purified from formalin fixed paraffin embedded tissues may routinely yield up to 50% intronic reads.
    5. The Epicentre TotalScript RNA-Seq Kit (Epicentre cat# TSRNA1296) is designed for an input of 1-5 ng of total RNA in a volume of 12 ul to a RNA sequencing library. Libraries constructed with this kit are directional (sequence reads occur in the same orientation as antisense RNA). This kit works best with RNA samples that have a RNA Integrity Number of 7.0 or higher. The kit does not include a polyA enrichment step and instead reduces rRNA representation using a proprietary cDNA synthesis buffer that results in reduced levels of rRNA in the sequence reads. However, these libraries may contain up to 25% rRNA. This kit works best with total RNA samples exhibiting a RIN number of 7 or higher.
    6. The NuGEN Ovation RNA-Seq System v2 (NuGen cat# 7102) provides a method for preparing amplified cDNA from 1-100 ng of total RNA in a volume of 8 ul. Amplified RNA generated by this kit can then be used to construct a non-stranded sequencing library. The protocol used with this kit enables both 3' representation of mRNA in addition to random priming throughout the RNA transcript. Representation of rRNA in the amplified cDNA is suppressed but approximately 15-20% of the sequence reads will likely align to rRNA. Following amplification, the cDNA is sheared with a Covaris AFA instrument and library construction is completed using the Illumina TruSeq Nano DNA Sample Prep kit.
  2. Purification of RNA: Total RNA should be purified using a column-based kit such as those by Qiagen (RNeasy Mini Kit; RNeasy Lipid Tissue Mini Kit; miRNeasy Mini Kit; RNeasy Plant Mini Kit), or the Zymo Research Direct-zol RNA MiniPrep Kit. In all cases, it is recommended to include on-column DNase treatment to minimize DNA contribution in the purified RNA sample.
  3. Purification of RNA from FFPE Tissues: Total RNA purified from FFPE tissues can be used for RNA-seq experiments when using a library preparation kit that includes Ribo-Zero. Kits that are recommended for the isolation of RNA from FFPE tissues includes the Ambion RecoverAll Total Nucleic Acid Purification System for FFPE and the Qiagen RNeasy FFPE Kit. In all cases, the RNA should be treated with DNase to minimize background sequence reads contributed by DNA. The Kapa Biosystems hgDNA Quantification and QC Kit can be used to perform quantitative assessment of the amount of genomic DNA remaining in the RNA sample. It is our observation that libraries constructed from RNA samples that were purified from formalin fixed paraffin embedded tissues may routinely include 50% intronic and 10-20% intergenic read contribution in RNA sequencing data.
  4. Intergenic Reads in RNA Sequencing Data: DNase treatment is essential for RNA samples in which sequencing libraries will be constructed that take advantage of rRNA removal by Ribo-Zero. Although on-column DNase treatment substantially reduces the quantity of contaminating DNA in the purified RNA sample, it does not fully remove contaminating DNA. It is our observation that RNA sequencing data may routinely contain 10-20% intergenic read contribution in the sequencing data of libraries constructed when removing rRNA with Ribo-Zero. In contrast, intergenic sequence reads are substantially depleted in libraries constructed following oligo(dT) selection of polyA RNA.
  5. Avoid Organic Extraction Methods: The use of organic extraction reagents, such as Trizol, as a stand-alone method for RNA purification is discouraged. Although cost is lower and yield may be a bit higher than column-based purification methods, the quality of RNA purified by these protocols tends to lower due to co-precipitation of contaminants during the EtOH precipitation step. Furthermore, organic contaminants that co-purify with the RNA can have an inhibitory step on enzymatic steps included in RNA-seq library construction and library prep of these samples may occasionally fail.
  6. Tissues with High Fat Content and certain embryonic tissues benefit from the inclusion of an organic step in RNA purification to remove excessive amounts of lipid and carbohydrate. We recommend purifying RNA from these tissues with a kit such as the Qiagen RNeasy Tissue Lipid Mini Kit, the Qiagen miRNeasy Mini Kit or the Zymo Research Direct-zol RNA MiniPrep Kit which include an organic extraction step prior to column purification. However, we advise against using an organic reagent such as Trizol as a stand-alone method for purification of RNA from these tissues.
  7. Ribo-Zero: RNA samples must be free of organics and salts (magnesium or guanidine) prior to ribo-zero treatment. Carry-over of these reagents can adversely affect removal of rRNA. Ribo-Zero can effectively remove rRNA from a number of different species and a species compatibility guide for the reagent can be found on the Epicentre website using the following link: http://www.epibio.com/applications/rna-sequencing/rrna-removal/ribo-zero-rrna-removal-kits-species-compatibility.
  8. 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. 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.
  9. RNA Fragmentation: RNA is chemically fragmented during library construction when using Illumina TruSeq kits. The fragmentation step results in an RNA-seq library that includes inserts that range from 100-400 bp. The mean insert size in an Illumina RNA sequencing library is approximately 160 bp. Fragmentation conditions can be modified for investigators that require longer inserts such as when sequencing the libraries on paired end runs.
  10. Library QC: Following construction of an RNA sequencing library, the library will be 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.