NGS Sample Prep

 

The core prepares libraries that are suitable for sequencing on Illumina sequencers. Library preparation includes Bioanalyzer and MiSeq quality control, normalization, pooling, and sample submission to UCSF sequencing cores. We offer several standard protocols for sample preparation. 

RNA-Seq

Illumina TruSeq Stranded mRNA: this method generates strand-specific libraries covering protein coding RNAs (mRNAs) and other long polyadenylated transcripts. This method requires 100 ng-1 µg total RNA starting material and a polyA selection step is included in the method. 

Illumina TruSeq Stranded Total RNA with Ribozero Gold: this method generates strand-specific libraries covering both polyadenylated and non-polyadenylated long transcripts. Requires 100 ng-1 µg total RNA and a ribosomal depletion step is used to remove rRNAs, which would otherwise dominate the libraries. We also offer a version of this method that includes globin depletion and is useful for blood samples.

NuGen Ovation/NexteraXT: these methods are appropriate when less RNA is available for analysis, and can be used with as little as 500 pg total RNA.

ChIP-Seq

NexteraXT: this method can start with as little as 1 ng of chromatin immuno-precipitated DNA (supplied by the user).

Small RNA-Seq

Modified Illumina TruSeq Small RNA: this method is designed to sequence miRNAs but also captures some other small RNAs and fragments of larger RNAs. We use random-end adapters to minimize ligation bias and optimized the method to work with as little as 10 ng of total RNA.