Figure 2

Overview of DEEPER-Capture procedure. (A) Overview of RNA probe production. Target DNA sequences are cloned into pcDNA 6.2 vector. Two clones for each target sequence are constructed, with a T7 promoter inserted upstream of the target sequence on either strand. For each DNA target sequence, in the “+” clone, T7 promoter is inserted at the 5′ end of the plus strand of the target sequence; and in the “−” clone, T7 promoter is inserted at the 5′ end of the minus strand of the target sequence. “+” clone and “−” clone for each target sequence are separated into 2 pools and are subject to in vitro transcription to produce biotin labeled RNA probes individually. 2 pools of RNA probes are then sheared into 100-150nt RNA fragments by ultrasonication. (B) Overview of DEEPER-Capture workflow. A hybridization mixture is prepared by first heated dissociation of double-stranded DNA molecules in the library, blocking primers and two pools of RNA probes are added into the system sequentially in a heated environment. After hybridization, streptavidin beads are used to isolate RNA probes and their hybridized DNA targets from the system. Target DNA sequences are then eluted from the beads and subject to PCR amplification and NGS sequencing.