The BioMicro Center supports a broad variety of standard library preparation methods for RNAseq. The choice of method is highly dependent on the type of input, the amount of input RNA available, and the quality of the input RNA. The key in all RNAseq methods is the avoidance of ribosomal RNA, which would dominate the library preparation. Below is a summary of the methods we utilize routinely in the core. For High-Throughput RNA library preparation, please see our page for methods designed specifically for large sample batches.
NEBNext Ultra II Directional RNA preparation with poly(A) selection is used to selectively capture mRNA from high-quality total RNA. The selection of mRNA transcripts occurs by hybridization of the poly(A)-tail to magnetic oligo-d(T) beads. We can reliably generate RNA-Seq libraries with inputs ranging from 10ng to 100ng (recommended input of > 50ng). High-quality samples are required because the method is reliant on poly(A)-tails during RNA isolation. We also offer a high-throughput protocol.
NEBNext Ultra II Directional RNA preparation with ribosomal RNA (rRNA) depletion is used to deplete rRNA by enzymatic degradation using single-stranded DNA probes that target rRNAs, leaving all other RNA species present and available for library preparation. This depletion method is agnostic to input sample quality and is the go-to method if samples don't meet quality requirements for poly(A) selection. This method is suited for inputs ranging from 5ng to 100ng (recommended input of > 50ng). Due to probe design used in the depletion, this method is currently only available for Human/Mouse/Rat samples. We also offer a high-throughput protocol.
The BioMicro Center utilizes the Kapa mRNA hyperprep kit for standard RNA libraries. This workflow is very similar to Illumina's TruSeq chemistry at lower cost and is streamlined for automation. This chemistry uses polyT beads to isolate the mRNA from the rRNA and tRNA. The use of these beads requires that the RNA be of very high quality or only the 3' end of transcripts will be isolated. Purified mRNA is then fragmented with metal and random priming is used to convert the sample to cDNA. Once double-stranded cDNA is generated, LMPCR is performed to create the indexed Illumina library. The BioMicro Center offers mRNA HyperPrep as a single sample reaction or in batches of 24 done on the TecanEvo 150s.
For samples with degraded RNA or samples where you are interested in looking at non-polyA RNAs, the BioMicro Center utilizes the Kapa RNA RiboErase and Illumina RiboZero for Human/Mouse/Rat samples. RiboErase uses RNAseH to degrade rRNAs while RiboZero uses magnetic beads coupled to rRNA sequences to remove these sequences from the solution. The remaining mRNA fragments can then be converted in to cDNA and prepared using the NEB Ultra II Directional RNA or Kapa mRNA Hyperprep kit to produce the Illumina library.
For samples with less then 50ng of input, the BioMicro Center utilizes the Clontech SMARTseq v4 system. This system differs from the TruSeq chemistry in that it begins with cDNA generation using polyT priming followed by strand switching oligos. The use of polyT priming requires the RNA to be of high quality. Full length double-stranded cDNAs are generated and amplified by PCR. These cDNAs are then prepared into Illumina libraries using the NexteraXT chemistry from Illumina. Data from this system is of similar quality to samples created with Illumina TruSeq chemistry but is not stranded. Single samples can be prepared by hand. Batches of 24, 96 or 384 samples can be prepared using the older SMARTseq v2 chemistry on the Mosquito HV resulting in significantly lower costs/sample.
For samples with less then 100ng of input and restricted input amounts, our kit of choice is the Clontech SMARTer Stranded Total RNAseq Kit - Pico Input -- or more simply, Clontech ZapR . This kit utilizes the same template switching as the v4 kit but uses random primers on fragmented RNA. The key is the ZapR enzyme which is used post library production to, in a targeted manner, cause breaks in Illumina library molecules that contain rRNA reads. These breaks make the rRNA containing molecules unreadable. Currently this chemistry is only available as single samples but we are working to adapt it to the Mosquito HV system.
In analyzing data from this kit, we have observed that the first few nucleotides from many reads appear to have a very high mismatch rate, particularly from low input samples or samples that possibly may not be as clean as desired. We believe this is a results of the template switching and random priming. A 5nt trim from the 5'end of the read can significantly improve data quality.
Additional Chemistries Available in the BioMicro Center
For some applications of RNAseq, such as splice choice determination, having a precise knowledge of the insert size is critical. While the SPRIworks does provide some size selection (typically restricting fragments to between 150 and 350bp), this can be too wide for some methodologies. In these cases, after libraries are amplified, they can be run on the Sage BluePippin (either singly or pooled). Here the size distribution can be much tighter, with most of the DNA fragments being within a 50nt range.
