Info Sheet: RNA-seq (low input)

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

Workflow

  1. QC: RNA quantification and checking the RNA integrity
  2. Library construction according to the RIN value of samples (RIN > 8 - PolyA enrichment; RIN < 8 universal priming and ribosomal depletion)
  3. Library QC
  4. Sequencing
  5. Bioinformatics data analysis (optional)
Expected turnaround time: 6-8 weeks after passing QC

Input requirements

  • at least 10 ng total RNA; volume: 20 ul, concentration must be 0.1 ng / ul - 1.25 ng /ul
  • RNA must be purified and DNA-free
  • 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

Read types (other configurations only on request):
NovaSeq (50 bp + 50 bp paired-end reads)
NextSeq500 (150bp single-end reads)
Recommended sequencing depth: Poly A enrichment - 20 M reads / sample; ribosomal depletion - 40 Mio reads / sample

Costs

Library prep (Poly A enrichment, RIN 8 or above) 92.5 CH / sample
Library prep (ribo depletion for degraded samples) 185 CHF/ sample
Sequencing 200 Mio reads (10 or 5 samples) 769.74 CHF
Bioinformatics (up to 15 samples, 3 comparisons): 800 CHF

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