Info Sheet: total RNA-seq
How to submit samples for Illumina/Short Read Sequencing.pdf
Workflow
- QC: RNA quantification and checking the RNA integrity
- Library construction from total RNA using ribo-depletion
- Library QC
- Sequencing
- Bioinformatics data analysis (optional)
Input requirements
- at least 300 ng total RNA + 3 ul for QC
- at least 15 μl sample volume
- concentration between 30 ng/μl - 100 ng/μl
- RNA must be DNA free (e.g. DNase treated)
- RNA from whole blood have special requirements (we must deplete for globin)!
- RNA should have RIN > 2, inquire for highly degraded RNA
Experimental design
3 or more replicates per condition (e.g. cell lines, mouse models, …)5 or more replicates per condition (e.g. humans, high variability, low quality RNA, elaborate/complex extraction)
Standard Sequencing Recommendations
NovaSeq; 100bp single-end reads; 40M reads/samplePacks of 200M reads
Costs for UZH and ETHZ research groups
Library preparation | 190 CHF / sample |
Sequencing 200M reads (5 samples) | 1037 CHF |
Bioinformatics (up to 15 samples, 3 comparisons) | 880 CHF |
Alternatives
mRNA-seq using poly-A selection
single-cell RNA-seq
ribosome profiling
higher sequencing depth
low input protocols (1 ng - 10 ng)
Back to Short Read/Illumina Sequencing
Back to FGCZ Main Page