Should I perform size selection of TeloPrime V2 cDNA for Iso-Seq™?
Size selection can be used to enhance the proportion of longer cDNAs in an Iso-Seq™ library for PacBio long-read sequencing. Size selection of TeloPrime V2 cDNA for Iso-Seq™ is optional. If you wish to perform size selection prior to Iso-Seq™ library prep please follow the guidelines in the Iso-Seq™ Protocol and use the Ampure® PB Beads (from PacBio) for all purification steps post-PCR. For the endpoint PCR for TeloPrime the following modifications are suggested so as to enable sufficient material for size selection and downstream library prep:
Size selection is optional for PacBio Iso-Seq™ library preparation. Size selection may enhance the proportion of longer transcripts that can be sequenced at higher depth during the run, but is not essential. TeloPrime V2 Iso-Seq™ libraries have been successfully sequenced with and without size selection. When performing size selection, please follow the instructions provided in the PacBio Iso-Seq™ protocol (https://www.pacb.com/wp-content/uploads/Procedure-Checklist-Iso-Seq-Express-Template-Preparation-for-Sequel-and-Sequel-II-Systems.pdf):
Size selection may not be necessary in the following cases:
Plant RNA – May have shorter average transcript length. Experienced customers report size selection is not necessary for plant samples.
When starting total RNA input quality is below RIN 8. Here the input is already more degraded and therefore less longer full-length cDNAs will be generated.
If normal transcript abundance should be preserved, as size selection alters the relative abundance of longer vs. shorter cDNAs.
If you do choose to perform size selection (to enrich longer cDNA fraction) please consider the following tips:
Prepare 2 – 4 endpoint PCRs when amplifying the TeloPrime cDNA – for each PCR use e.g. 2 – 4 µl of cDNA as template and adjust PCR cycle number accordingly. A higher starting yield of cDNA is needed for size selection due to re-pooling of the larger fraction at equimolar level to the shorter fraction. Each individual PCR should produce >600 ng of cDNA if PCR cycles are correctly optimized using the qPCR assay.
Pool the PCR products and adjust the total volume to 160 µl with molecular biology grade water.
Take 100 µl of this pool (10 volumes) and purify with 0.4x or 0.5x Ampure® PB beads according to the PacBio IsoSeq protocol.
Take 60 µl of this pool (6 volumes) and purify with 1x Ampure® PB beads according to the Pacbio IsoSeq protocol.
Perform QC on each fraction and re-pool the cDNA in equimolar manner according to PacBio protocol and continue with SMRT bell ligation.