Guidelines for bulk RNA Sequencing
GENERAL GUIDELINES FOR SAMPLE PREPARATION
There are two workflows for bulk RNA-Sequencing: so-called mRNA Seq targeting polyadenylated mRNAs and total RNA-Seq, where ribosomal RNA is depleted using species-specific rRNA hybridization probes.
The requirements of input per sample differ between both workflows and according to the RNA quality used.
mRNA-Seq
mRNA-Seq uses poly-dT oligo based enrichment of polyadenylated RNA and is the most frequently applied workflow; for this we require good quality, non-fragmented RNA (RIN values of at least 7, but >9 for optimal quality (see section [Sample quality control and quantification]).
Total RNA-Seq?
Total RNA-Seq using rRNA depletion by probe-based hybridization is suitable for samples with lower RNA integrity (RIN < 7) or when detecting various RNA forms beyond polyadenylated RNAs is desired.
Maintaining not fully processed transcripts in total RNA-Seq can lead to more intronic reads compared to mRNA-Seq, hence requiring sequencing of approximately 20% more reads. This adjustment helps capture a broader range of RNA species and ensures comprehensive coverage of the transcriptome.
CHECKLIST CRITICAL POINTS
INPUT MATERIAL
Type
The required input is total RNA, purified and eluted in sterilized nuclease-free water. For the total RNA-Seq workflow the RNA should be DNase treated.
* We do not offer the service of RNA extraction, but we are glad to advise on best practices for RNA extraction.
Amount
Ideal = 300-1000ng (minimum 50ng; preferred 500ng)
Volume
mRNA-Seq: max. volume = 50µl
total RNA-Seq: max.volume = 11µl
* Please elute samples in nuclease-free water (NFW). Do not use DEPC water
Sample quantification and quality control
For quantification we recommend using Qubit (Thermofisher Scientific) or Bioanalyzer (Agilent Technologies). We don’t accept Nanodrop measurements, as they are not accurate.
We recommend to check RNA integrity and quantify samples with a fragment analyser (e.g. Bioanalyzer from Agilent Technologies) to determine the RIN value (Schroeder et al. 2006). For mRNA-Seq the RIN values should be >7. For total RNA-Seq we accept samples with RIN >5. We don’t provide RNA QC services, so we cannot take responsibility for failed sequencing runs of samples submitted without proper quality control.
** please enter the RIN values of each sample in the “notes” field when submitting samples via the Rosalind Client Portal.
PREPARING ALIQUOTS FOR SEQUENCING
Extracted total RNA should be aliquoted with identical concentrations and volume. In case of few lower concentrated outliers, we recommend to not scale all remaining samples down; rather please aliquot equal amounts of the higher-concentrated samples and inform us about the outliers.
Supported plastics for sample submission
Samples can only be submitted in 96-well plates, 384-well plates or 8-strip PCR tubes with single lids. Strips or plates that are fully compatible with our systems are Sarstedt strips (#72.991.002), Eppendorf 96-well twin.tec® skirted PCR plates (e.g. Cat.no. 0030128648) and 4titude 384-well FrameStar plates (Cat. no. 4ti-LB0384/C). These can be provided upon request. When submitting in plates please fill them column-wise w/o empty wells in between.
Please identify tubes/samples with clear and short labels, e.g. with the sample ID (created in Rosalind during online sample submission) or a running index clearly referenced under the corresponding samples submitted over the Rosalind Client Portal.
SEQUENCING
Standard sequencing for RNA-Seq is paired-end 2 x 100bp. Different read lengths can be requested. Our workflow provides strand-specific sequencing data.
We recommend a sequencing depth of 40 mio read pairs/sample for mRNA-Seq and 50 mio read pairs / sample for total RNA-Seq. Specificities of the species, sample, or project design, can influence the optimal sequencing coverage. We are glad to provide guidance with this.
BIOINFORMATICS ANALYSIS
At least 3 biological replicates are required for proper data analysis. Factors that influence the number of replicates required for a decent statistical analysis are explained in the infobox below.
We have an established pipeline that generates both quantitative and qualitative information about the samples. The workflow comprises all steps from initial quality control, read alignment and read count, correction for batch effects, sample correlation, cluster analyses (e.g. PCA), visualizations of variable and highly expressed genes, and differential gene expression analyses.
Please be aware that the number of samples per experimental group or condition in gene expression analyses are influenced by the following factors:
Biological and technical variation of replicates
The less homogeneous your biological replicates are, the more are you limited in finding statistical significantly expressed genes. This applies in particular to small differences in gene expression. Therefore rule of thumb: the smaller the differences you expext or the higher the sample to sample variability the more replicates you should do per experimental group.
Heterogeneity of sample quality
Differences in RNA integrity can introduce technical noise, thereby diminishing the sensitivity of detecting differentially expressed genes (DEGs). For bulk RNA-Seq we advise utilizing the total RNA-Seq workflow for samples of lower quality or greater heterogeneity. This approach is advantageous as it is not reliant on intact poly-A tails of mRNA, ensuring robustness in such challenging sample conditions.
SAMPLE SUBMISSION
Please log in to Rosalind to enter all necessary project information making use of the “Project Description” field during the project set-up dialogue. Give all relevant details about project design, sample preparation method and how the samples are eluted and/or preserved. Sample-specific information such as concentration, volume, used preparation protocols etc. should be provided when the samples are added to the created project.
Please also indicate if you require a bioinformatics analysis during project setup.
Submission of large sample batches
The Rosalind customer portal offers an Excel-based alternative for sample submission of more than 30 samples delivered in either 8 well strips, 96- or 384-well plates. You can specify these supported formats in the portal’s sample submission dialog and download a formatted Excel template for entering your sample details. Plate identifiers and well coordinates are pre-set to facilitate easy data entry. Once completed, please email the Excel file to genomecenter@tu-dresden.de. Do not upload individual samples to Rosalind in this case.
To submit samples, you must be registered in our Rosalind client portal. Therefore, please send an e-mail to genomecenter@tu-dresden.de with your contact details (name, working group, working group leader, your e-mail address and telephone number). This will enable us to set up an account for you. After you have received an automatic e-mail, please log in to Rosalind with your login details. If needed, we can reset your login details.
Delivering or shipping of samples
Once the project and sample information has been entered into the Rosalind Client Portal, please put the samples in a small plastic bag labeled with your name and Rosalind project ID.
Samples can be personally delivered to the lab at the (CRTD, 3rd floor south), daily from 9-12h and 13-16h. If needed, contact us to deliver samples outside of this time window (+49 351 458 82362 or email us at genomecenter@tu-dresden.de).
Please deliver samples under suitable conditions! When shipping your samples, please use an adequate-sized box and ensure a proper amount of cooling material to hold the sample temperature until it reaches us. The package should be addressed to:
DRESDEN-concept Genome Center
c/o Center for Regenerative Therapies Dresden
Technische Universität Dresden
Fetscherstraße 105
01307 Dresden
RNA should be delivered with a sufficient amount of dry ice to avoid degradation. If you need to work with very low RNA concentrations, please get in touch with us in advance to discuss the best strategy to deliver your samples to our lab under optimal conditions.
When submitting in plates please also get in touch with us to select the right sealing material for tight sealing of your plates!
Following up project progress
You can accompany the status of your project in our Rosalind Client Portal. Documents with information about the preparation method and quality control of samples and libraries are provided for your project.