RNA-Sequencing (high-throughput mRNA-Seq, total RNA-seq, 3′-Tag-Seq, miRNA-seq, … )

RNA-Sequencing Libraries

Something of a misnomer because all the libraries end up as DNA, but this refers to the starting material. We offer RNA-seq library preparation, with multiple options such as ribo-depletion, poly-A enrichment, 3′-Tag-Seq (QuantSeq) libraries as described below as well as micro-RNA (miRNA) and small RNA library preps.  All our library preps are strand-specific.

Please see the Comprehensive Sample Requirements Page and consult our FAQs for technical questions

Guidelines for Submission of Library-Worthy RNA
Provide at least 1 ug (2 ug preferred) of total RNA at a concentration of at least 50 ng/ul (1 ug for Poly-A enrichment; 2 ug for ribo-depletion libraries; using less starting material is possible, but we can’t guarantee results). Please make sure that your RNA isolation protocol employs a DNAse digestion step or other means to remove DNA from the sample. On an agarose gel, DNA contamination will be visible as a smear of band of fragments considerably larger than the RNA (>10 kb).  On the Bioanalyzer RNA-chips DNA will be visible in the size range from 4 to 10 kb.  To verify the purity of the RNA samples the 260/280 ratio should be between 1.8 and 2.1 and 260/230 ratio should be higher than 1.5.  Poly-A enrichment, ribo-depletion and strand specific library prep are among the commonly requested types of service (more technical details on this appear below).  If the RNA quality allows poly-A enrichment is the first choice. Libraries for slightly degraded RNA samples should be prepared using ribo-depletion protocols.  Bacterial RNA-seq will always require ribo-depletion.  If possible please avoid RNA extraction protocols involving Trizol or related phenol containing reagents (silica column based kits are less likely to retain contaminants). If using Trizol, protocols that contain a column based cleanup (e.g. Direct-zol, TRIzolPlus) are recommended.  Please note that an additional column cleanup is mandatory for RNA isolated from blood sample PAXgene or Tempus tubes (for blood sample preservation)  or with the accompanying PAXgene and Tempus RNA isolation kits. RNA samples should be eluted in molecular biology grade water, always stored in a -80 degree freezer and shipped on dry ice.
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All RNA samples require a Bioanalyzer sample QC (or equivalent).  Such QC traces can be submitted by the customers or we can run the QC for a fee instead.
All RNA samples need to be DNA-free.

RNA-Seq after Poly-A Enrichment
Total RNA samples can contain up to 90% ribosomal RNA (rRNA) sequences, which are uninformative for transcriptome or gene expression studies, while mRNAs typically make up only 1 to 2% of total RNA. Thus the enrichment of samples for mRNAs is highly desirable. Positive selection via poly-A enrichment is the most commonly used method to enrich mRNA sequences from eukaryotic total RNA samples; mRNAs are selected by hybridization to poly-T oligos bound to magnetic beads.  This method generates the highest percentage of reads mapping to protein encoding genes and thus is the first choice for most applications.  Poly-A enrichment however requires high-quality total RNA samples.  We suggest following the recommendations from Illumina – for human/animal samples use total RNA with a bioanalyzer RIN score of 8 or better, for plant material RIN numbers can be lower and tissue-specific (this is mainly a function of the chloroplast content) but should generally be higher than 7.

RNA-Seq after Ribosomal RNA Depletion
Ribosomal RNA depletion is negative selection method to avoid sequencing the dominant rRNA content.  There are multiple kits commercially available to remove ribosomal RNAs (rRNA) from your total RNA. Ribosomal RNA depletion is applied when transcripts do not carry polyA (bacterial RNA), when you desire to retain all long non-coding RNA (lncRNA) and polyA classes of RNA in your sample, and also for lower quality RNA samples. Commercial kits containing rRNA removal solution are available for different types of total RNA; they include human, mouse, rat, bacteria (gram positive or negative), plant leaf, plant seed and root, and yeast. Ribo depletion protocols can further enable the analysis of slightly degraded RNA samples (the RIN scores should nevertheless best be 5 or higher). We ask for at least 1 ug of total RNA for the preparation ribo-depleted libraries. As always libraries can be generated from less material, but the complexity can suffer.

3′-Tag-Seq (QuantSeq) Gene Expression Profiling
3’-Tag-Seq is a protocol to generate low-cost and low-noise gene expression profiling data. The protocol is less dependent on RNA sample integrity than poly-A enrichment protocols. More than 48 samples can be sequenced per lane. Please see this FAQ for detailed information as well as the 3’-Tag-Seq page.  For high-throughput 3’Tag-Seq library generation we require pure total RNA samples at a concentration of 100 ng/ul.  For custom 3’-Tag-Seq library preps the input amounts can be a low as 10 ng total. The RNA samples for this protocol need to be isolated or cleaned-up by spin-column protocols.  3-Tag-Seq libraries are sequenced by single-end sequencing on the HiSeq 4000 or the NextSeq.

Micro RNA and Small RNA Libraries
We offer library construction for micro and small RNAs from total RNA using the Illumina protocol and reagents. We size select the libraries with high precision using the Blue Pippin system. The minimum recommended amount of total RNA required for these preps is 100 ng (recommendations for humans samples). Since the total RNA composition can vary widely between tissues and organisms, please aim to provide at least 1 ug of total RNA. Please also take care that you RNA isolation method actually retains micro and small RNAs.  The total RNA samples should be submitted in molecular biology grade water at a concentration of 200 ng/ul.  High quality RNA is recommended (the total RNA samples should have RIN scores of 8 or higher according to a Bioanalyzer QC) and should have been DNAse treated before sample submission.
We are using the NEXTflex™ Small RNA-Seq kit for the generation of micro RNA and small RNA-seq libraries because it significantly reduces sequence-specific biases during library preparation by employing adapters with randomized ligation junctions.  For most applications, the randomized bases should be trimmed before before mapping the reads.

Strand-Specific RNA Libraries
By default we always generate strand-specific RNA-seq libraries. Please let us know if you would prefer the traditional non-stranded library prep instead. Strand-specific (also known as stranded or directional) RNA-seq libraries substantially enhance the value of an RNA-seq experiment. They add information on the originating strand and thus can precisely delineate the boundaries of transcripts in regions with genes on opposite strands, and can determine the transcribed strand of non-coding RNAs. During the cDNA synthesis dUTP is incorporated in the second-stand synthesis. After adapter ligation the dUTP-containing strand is selectively degraded, to preserve strand information for RNA-seq. The forward read of the resulting sequencing data thus represents the “anti-sense strand” and the reverse read the “sense strand” of the genes (for Trinity transcriptome assemblies the “–RF” orientation flag should be used).

Other Library Considerations

Library Indexing and Pooling
Indexing, also called barcoding, allows for the sequencing of multiple libraries in a single lane, i.e., multiplexing. By default all libraries generated by us have a barcode. Multiplexing is required when the typical lane output of 15-25 million reads from the MiSeq, 300-400 million reads from the HiSeq 4000 is greater than required for a single library (e.g., in sequencing BACs, PCR generated fragments, small microbial genomes, transcriptomes, exome, ChIP, and small RNA applications). Multiplexing is also the best way to minimize potential lane-to-lane sequencing variation, as all of your samples are subject to the same sequencing conditions. For example, if you require two sequencing lanes for six samples we recommend 6-plexing and sequencing over two lanes, instead of 3-plexing per lane. The principle is that short nucleotide “barcodes” are appended to each library using specific adapters containing those sequences. Libraries containing different indexed adapters are then constructed, quantified, pooled in equimolar amounts, and sequenced. Deconvoluting the barcodes informatically allows multiple libraries to be sequenced in a single lane at a potential cost and time saving. To date, two methods have been exploited for this: using the commercially available indexing kits (Illumina TruSeq, Nextera, or Bioo Scientific) or synthesizing your own adapter oligos with your own barcodes.   Bioo Scientific offers Illumina-compatible barcodes (NEXTflex) with up to 384 barcodes. The Nextera kit (Epicentre/Illumina) uses dual indexing and transposon mediated fragmentation (‘tagmentation’) followed by PCR amplification to integrate barcoded adapters (so a PCR-free library is not an option using the Nextera kit). The dual indexing/adapter tagging strategy (with up to 12 indices available for adapter 1 and up to eight indices for adapter 2) permits up to 96 unique dual index combinations.
When sequencing on the HiSeq 4000 and especially on the NovaSeq it is highly recommended to use uniquely-dual-indexed adapters (UDI adapters) to avoid index hopping artifacts. Please see this FAQ.

Buffer Compositions

Common buffers for DNA and RNA samples are:
Molecular Biology grade water (RNAse free, but not DEPC treated)
EB-Buffer: 10mM TRIS (pH= 8.0-8.4) – e.g. Qiagen EB Buffer
EBT-Buffer: 10mM TRIS, 0,1%Tween20 (pH=8.0-8.4)
TE-Buffer: 10mM TRIS, 1 mM EDTA (pH=8.0-8.4)
TLE-Buffer: 10mM TRIS, 0.1 mM EDTA (pH=8.0-8.4)

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