Profiling of RNA-binding protein LSM14B target sites in oocyte by LACE-seq

RNA-binding proteins (RBPs) have essential functions during oocyte development. In order to explore the regulatory role of RNA-binding protein LSM14B in oocyte, we determined the binding sites and regulatory mechanisms for LSM14B by LACE-seq. Cells are first irradiated with ultraviolet C (UV-C) light to crosslink RBPs and their interacting RNAs. We next use LSM14B antibody to specifically pull-down protein–RNA complexes from the cell lysate. To cut RNAs into individual RBP-associated short fragments, we treat the RBP–RNA complexes on the beads with micrococcal nuclease (MNase). The 3′ ends of fragmented RNAs are then dephosphorylated and ligated with a 5′ pre-adenylated linker containing four randomized nucleotides. Next, a biotinylated primer containing the T7 promoter is used for reverse transcription on beads. Since reverse transcriptase would efficiently stop before the intact RBP–RNA crosslinking sites, sequencing these truncated cDNA ends enables us to identify 3′ sequences downstream of the bound element of a defined RBP. As immunopurified RNAs from single or dozens of cells are very limited, we adopted a T7 RNA polymerase-based in vitro transcription (IVT) approach to amplify trace amounts of truncated cDNAs linearly. The cDNAs are tailed with poly(A) and subsequently enriched using streptavidin beads via the biotin moiety at the 5′ end of the T7 primer. Next, we synthesize second-strand cDNA on beads with an adaptor containing oligo-(dT). After pre-amplifying the double-stranded DNA (dsDNA) fragments by PCR for 14–18 cycles, the products are purified and used for IVT. Subsequently, the transcribed RNAs are converted into libraries for single-end deep sequencing (~180bp).