Info Sheet: mRNA-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 poly-A selection
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 20 μl sample volume. We will use at least 2ul for QC!
• concentration between 20 ng/μl - 100 ng/μl
• Bacterial RNA: 5 ng of ribosomal RNA depleted samples in 5 μl volume
• RNA from whole blood must be depleted from Globin first!
• RNA must be DNA free (e.g. DNase treated)
• RNA must have RIN > 8 (will be checked by FGCZ)

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; 20-25M reads/sample
Packs of 200M reads

Costs for UZH and ETHZ research groups

Library preparation	124 CHF / sample
Sequencing 200M reads (8-10 samples)	1037 CHF
Bioinformatics package	880 CHF

Alternatives
total RNA-seq using ribosomal depletion
single-cell RNA-seq
higher sequencing depth
low input protocols (1 ng - 100 ng)

Back to Short Read/Illumina Sequencing
Back to FGCZ Main Page