Dear DNA Technologies Core Users,
We are happy to announce a new service for the new year: Batch-Tag-Seq Gene Expression Profiling. This service is designed to simplify both the planning and the analysis of gene expression profiling studies.
Batch-Tag-Seq is offered as packages that include both, library preps and sequencing, as well as optionally also data-analysis. Batch-Tag-Seq combines the simplicity and low-cost of microarray-based gene expression profiling with the advantages of high throughput sequencing: higher precision (low background and much higher dynamic range) and the ability to study unknown transcripts (no need to design species-specific arrays). Please see the details below.
We have now added the Qubit fluorometer to our array of shared instruments that are accessible to everybody on campus (after a short training).
Please let us know if we can help with any questions.
Batch-Tag-Seq – Gene Expression Profiling
3’-Tag-Seq is a protocol to generate exceptionally low-noise and low-cost gene expression profiling data. For more details on the technology please see our FAQs on 3′-Tag-RNA-Seq. We now offer Batch-Tag-Seq packages that include 3′-Tag-RNA-Seq library preparations & sequencing & optional data analysis at low per-sample rates. The differential gene expression (DGE) data analysis is performed by the Bioinformatics Core. For Batch-Tag-Seq we will collect samples and process them in larger batches and sequence the barcoded libraries together on sequencing runs, allowing us to offer affordable recharge rates on a per-sample basis. This also simplifies the budgeting and planning of experiments since scientists will not have to adjust their experiments to the sequencing capacity of the sequencers. Each Batch-Tag-Seq project requires a minimum of 12 samples.
Please note: In order to enable low cost DGE data analyses and short turnaround times, we will process the Batch-Tag-Seq samples in high throughput fashion. We will not spend time customizing the protocol for individual samples (for example we will not run sample cleanups, sample concentrations, or repeat the library preps or PCR amplification with varying cycle numbers). The 3′-Tag-RNA-Seq library prep protocol is very robust, therefore no problems should be expected as long as the RNA samples fulfill the sample requirements. Please note that the customer is responsible for the sample quality.
Further considerations:
– 3′-Tag-RNA-Seq is only suitable for eukaryotic total RNA-samples (A-tailed transcripts).
– For each condition at least 3 biological replicate samples need to be sequenced to allow for a meaningful DGE analysis. As with any other DGE study, statistically meaningful experiments might require higher replicate numbers, depending on the nature of the samples and the phenotypes. Both, the DNA Tech Core and the Bioinformatics Core (bioinformatics.core@ucdavis.edu), offer free consultations to discuss such details before starting projects.
– We require 15 ul of each total RNA sample at a concentration of 50 to 100 ng/ul for batch-processing (dnatech.genomecenter.ucdavis.edu/sample-requirements/).
– The RNA samples need to be dissolved in molecular biology grade H2O or EB buffer. As always RNA-seq samples need to be DNA-free.
– The sample concentration must be determined by fluorometry (e.g. Qubit; plate-reader with Ribo-Green), as spectrometry quantifications (e.g. Nanodrop) are very unreliable.
– To assure the chemical purity of the samples the absorbance ratios should be between 1.8 and 2.1 (260/280 nm ratio) and above 1.5 (260/230 nm ratio).
– If RNA-isolation protocols involving TriZol are used, the RNA should then be purified via a spin column kits (e.g. Zymo RNA clean & concentrate) to remove any solvent traces.
– 3′-Tag-Seq has a relatively high tolerance for RNA integrity variation. Nevertheless, we do not recommend using RNA samples with a RIN-score lower than 6 for batch processing. We cannot guarantee for the outcome of the library prep and the data for lower quality samples or samples without Bioanalyzer QC.
– The customers should provide Bioanalyzer traces (or equivalent). Alternatively we can also run the RNA sample QC on the Bioanalyzer or the LabChip GX for an additional fee.
– The libraries will be sequenced on Illumina HiSeq 4000 or NextSeq 500 sequencers with single-end 80 or 90 bp reads (SE84 or SE90). Please note that for some analysis pipelines it is recommended to trim off the first 11 bases from the reads. We will provide the full length data. Trimming is not necessary if you are using a local aligner (like STAR or BBmap). The sequences can be trimmed easily, for example with the “reformat” command from BBTools.
– You will receive 3 to 6 million reads per sample. For typical experiments about 2 million reads per sample are required for the DGE analysis of highly- and medium-expressed genes.
– The availability of a well annotated reference genome (including UTRs; to be provided by the customer) is a prerequisite for bioinformatic data analysis. The deliverables will include data QC, read-counts-per-gene tables, and DGE analysis for simple comparisons.
Custom 3’-Tag RNA-Seq: In contrast to the batch processing described here we can adjust the library prep parameters for 3’-Tag-Seq when running custom library preps. The custom protocols can generate usable data for inputs as low as 10 ng total and also work with degraded RNA samples. Custom protocols do require additional labor. Please inquire with us with a description of your samples.
