Transcriptome (Poly-A mRNA) sequencing. Our most common RNA-seq service is sequencing poly-A mRNA molecules for gene expression profiling and discovery. Researchers need only isolate the total RNA from their samples and ensure that the RNA is of high quality (RNA Integrity Number >= 8 is required) and free of DNA. We will use poly-dT magnetic beads to extract polyadenylated mRNAs and then make Illumina compatible cDNA sequencing libraries using a kit such as the Illumina TruSeq Stranded mRNA library kit. The minimum sample requirement is 100 ng of total RNA, but 1-4 micrograms is recommended. Up to 24 different samples can be combined. For gene expression profiling, we recommend ~ 30-50 million reads per sample (8-12 samples/NextSeq high capacity cartridge). However, more sequencing may be required if the genes of interest are not highly represented. Single sequencing reads, i.e. 1 x 75 bp, can be sufficient for profiling commonly expressed genes with high quality reference genomes. But, paired-end sequencing, i.e. 2 x 75 bp, is required for discovery projects where alternative transcripts or gene fusions are important. Paired-end sequencing may also be necessary when working with lower quality reference genomes.
Whole transcriptome sequencing (human/mouse/rat). If noncoding mRNA molecules are also of interest, then we can prepare whole transcriptome libraries containing both polyadenylated and non-polyadenylated mRNAs. Starting from customer-supplied total RNA, we will deplete the unwanted rRNA molecules from the sample using magnetic beads containing rRNA-binding primers (Ribo-Zero) before making the cDNA sequencing library. However, the Ribo-Zero rRNA depletion step is not as efficient in removing rRNA and so additional sequencing reads may be required.
Bacterial transcriptome sequencing. RNA-seq from bacterial RNA requires ribosomal RNA depletion similar to whole transcriptome sequencing. Ribo-Zero rRNA Removal kits are available for either gram-negative, gram-positive, or mixed populations of bacteria. Check the species compatibility tables for assistance in selecting the optimum Ribo-Zero kit.
Degraded RNA samples (FFPE). Partially degraded RNA samples are not suitable for poly-A mRNA enrichment. Instead, they need to be processed as whole transcriptome libraries using ribosomal depletion.
Small RNA sequencing. microRNA and other small RNAs are profiled using a different library preparation protocol than mRNAs, so small RNAs are excluded from the above transcriptome sequencing methods. We prepare small RNA libraries by including a gel electrophoresis size selection step on our Pippen Prep System (3% agarose cartridge). Up to 48 different index tags are available if the Illumina TruSeq Small RNA Library Prep Kit is used. Whole transcriptome amplification. When sample size is limiting, it may be impractical or impossible to obtain the minimum amount of total RNA (100 ng) required for a conventional cDNA library preparation. Then, a whole transcriptome amplification step is required to produce enough material for next-gen sequencing. We use the Qiagen REPLI-g Single Cell RNA Library Kit when this is necessary. This kit uses multiple displacement amplification (MDA) in a single tube protocol which is supposed to provide accurate amplification of all transcripts with negligible sequence bias. For previously purified total RNA, the input can range from 50 pg to 100 ng.