What method should I use to prepare cDNA generated with SMARTer Ultra low kits for sequencing?
What method should I use to prepare cDNA generated with SMARTer Ultra low kits for sequencing?
We recommend two preparation methods for Illumina sequencing platforms:
Covaris shearing followed by library construction with the ThruPLEX DNA-Seq Kit (Cat. # R400674–R400677). This kit is compatible with 50 pg–50 ng of fragmented, double-stranded DNA (<1,000 bp), allows multiplexing, and has been validated for downstream Illumina sequencing platforms.
The Nextera® XT DNA Sample Preparation Kit (Illumina, Cat. # FC-131-1024). We have found that 100–150 pg input cDNA from the SMARTer Ultra low kits gives optimal results with this sample preparation kit.
For the Ion Torrent sequencing platform, we recommend using the Ion Xpress Plus Fragment Library Preparation Kit (Thermo Fisher Scientific, Cat. # 4471269) and an Ion Xpress Barcode Adapter kit (Life Technologies, several Cat. #s.). This method is compatible with 1–10 ng of cDNA digested with AfaI (to remove SMART adapters) and enzymatically fragmented using reagents from the Ion Xpress Plus Fragment Library Preparation Kit.
Note: Ion Torrent library preparation is only compatible with cDNA generated using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing or the SMARTer Ultra Low Input RNA Kit for Sequencing - v3.