Info Sheet: total RNA-seq

How to submit samples for Illumina/Short Read Sequencing.pdf

Workflow

1. QC: RNA quantification and checking the RNA integrity
2. Library construction from total RNA using ribo-depletion
3. Library QC
4. Sequencing
5. Bioinformatics data analysis (optional)

Expected turnaround time: 6 weeks after passing QC

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

1.5 ml tubes labeled with B-Fabric Tube ID

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/sample
Packs 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)


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