Experimental design considerations
For genome annotation: up to 12-plex trancriptome can be sequenced on one 8M SMRT cell
For deep trancriptome profiling: one human transcriptome sequenced on one 8M SMRT cell.
RNA sample requirements
Both integrity and purity of total RNA used for Iso-Seq library prep should be evaluated before sample submission. :
- Sample integrity should be assessed by measuring RIN number using a Bioanalyzer 2100 instrument (Agilent Technology).
- RNA sample should have a RIN number ≥7.0 (ideally ≥8.0). Please note that RIN number is not possible to obtain for some species, in that case, please check for degradation products.
- Sample purity should be assessed using a Nanodrop spectrophotometer (Thermo Scientific).
- RNA sample should have an OD260/OD280 ratio ~2.0 and an OD260/OD230 ratio ≥2.0.
Other recommendations during isolation of total RNA for Iso-Seq library prep:
- Only use RNase-free water supplied un the reagent kit or other suppliers
- RNA sample should not be exposed to high temperatures or pH extremes (<6 or >9)
- RNA should not contain denaturants (e.g., guanidinium salts or phenol), detergents (e.g., SDS or Triton‐X100) or carryover contamination from the original organism.
RNA kit options
The product below have not been tested or validated by PacBio R&D.
Total RNA isolation: Qiagen RNeasy Plus kit, Sigma Spectrum Plant Total RNA kit, iNtRON Easy Spin Total RNA, TRIzol Reagent can be used to isolate total RNA from tissues and cells, including lipid-rich and difficult samples.
Sample delivery
Please deliver 20 µl of total RNA normalized to 43 ng/µl (in total, 860 ng).
NOTE: RNA samples should only be shipped with dry ice.
Data delivery
By default, we perform Iso-Seq analyses, with or without mapping to (customer provided) reference genome. All data from the analyses will be delivered together with raw data.
