Generating the Data
General Rules for Sample Preparation
Ignoring these simple guidelines will greatly increase the chances that your data will be unanalysable and/or your experiment unpublishable.
- Prepare all samples at the same time or as close as possible. The same person should prepare all samples
- Do not prepare “experiment” and “control” samples on different days or by different people. (Batch effects).
- Use high quality means to determine sample quality (RNA Integrity Number) (RIN >0.8) and quantity, and size (Tapestation, Qibit, Bioanalyzer)
- Don’t assume everything will work the first time (do pilot experiments) or every time (prepare extra samples)
Sample Amounts Guidelines
RNA-Seq Sample Recommendations (CCBR)
Sequencing
Illumina Sequencing Platforms
By far the most popular platforms for RNASEQ experiments are the Illumina family of sequencers. All are Sequencing by Synthesis (SbS) and produce Short read lengths (50 to 300 bp).
Consult with the Sequencing Core as to which is most appropriate for your experiment. The appropriate selection will be driven by cost, precision, speed, number of samples and number of reads required
Long Read Sequencing Platforms
Long read platforms by PacBio and Oxford Nanopore are beconing more popular and offer significant advantages over short read technologies in certain circumstances.