RNA Seq and Transcriptomics

Centrillion offers RNA Seq and transcriptomic analysis for both eukaryotic and prokaryotic samples. We help you determine the required coverage, identify the number of samples needed, advise on or perform RNA isolation, prepare libraries, and perform sequencing and bioinformatics analysis. Laboratory analytics are carried out throughout isolation and library preparation biochemistry steps to ensure that high quality data is produced.

Challenges

Getting exceptional results for transcriptome studies is dependent on good quality total RNA, and efficient depletion of rRNAs. Centrillion’s scientists are experts at handling RNA and will work closely with you to quality check your total RNA providing quality metrics for review. Accurate quantification of RNAs is essential to optimizing cluster density and maximizing total sequence yield.

Work Flow

RNA-Seq and transcriptome projects begin with total RNA extraction usually followed by library construction. Depending on the type and quality of your starting material, the preparation steps may vary. Generally total RNA is isolated and then rRNA depletion is carried out. cDNA is made from mRNA, adaptors annealed, libraries pooled and sequenced. High quality sequence data is aligned to reference data and comparisons between samples e.g. controls vs. cases, are carried out. See work flow below.

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Sample QC

Nucleic acid input requirements
– Minimum 1ug at a RIN of >8 for eukaryotic samples
– Consistent RIN among samples (within 1.5 is optimal)
– Concentration of 20ng/ul, < 55ul total volume

QC methods
– Bioanalyzer for quality and Bioanalyzer RIN score

RNA Library Prep

Total RNA
mRNA Enrichment
rRNA depletion
or polyA mRNA isolation
Fragmentation
cDNA Synthesis
End Repair, A tailing
Size Adapter Ligation
PCR Amplification
QC Library

Library Quality and Quantity Control

Library QC methods
– BioAnalyzer® (Agilent): Fragment size
– qPCR (Kapa®): Concentration, equimolar pooling
Or
– ddPCR (Biorad): Fragment size and quantity

Library QC pass criteria
– Min of 4nM
– Fragment size range

Sequencing

Available platforms
– HiSeq 2500
– MiSeq
Coverage depth in millions of reads
– 25M, 50M, 100M, custom
Read length
– HiSeq 2500 – 100bp paired ends, custom
– MiSeq – 50,150,300,500,or 600 cycles
Sequencing quality
– >80% of bases above Q30

Analysis and Delivery

Analysis
– Align reads to reference genome
– Assemble transcripts and compare to reference transcriptome
– Identify novel transcripts
– Determine relative expression level
– Provide RPKM FPKM values if requested
– Compare expression levels between samples if applicable
Delivery
– FTP or external data drive
– Data storage < 30 days after initial data delivery

To discuss your project contact us.