Profiling the binding sites of RNA-binding protein by LACE-seq


 RNA-binding proteins (RBPs) directly interact with various RNAs in living cells to regulate their processing, translation, and stability. Identifying the precise binding sites of RBPs is critical for appreciating their physiological or pathological roles in germline and early embryo development. Current methods typically need millions of cells to map RBP binding positions, which prevents us from appreciating the crucial role of RBPs in early development. Here, we present the LACE-seq method for unbiased mapping of RBP-binding sites at single-nucleotide resolution in fewer cells or even single oocytes. LACE-seq depends on RBP-mediated reverse transcription termination, and linear amplification of the cDNA ends for deep sequencing. To further promote its application, we describe a step-by-step protocol about how to construct a successful LACE-seq library.


Introduction
The human genome encodes over 1,500 RNA-binding proteins (RBPs) that play crucial roles in the development or differentiation of somatic cells, germ cells, and early embryonic cells [1][2][3] . RBPs achieve such regulatory functions by directly interacting with various target RNAs via speci c motifs or structural elements 1 . As a well-accepted working rule, RBPs usually regulate gene expressions in a positiondependent manner 4 . For example, the preferable bindings of RBPs at intronic regions usually suggest potential regulations in alternative splicing, while their bindings at 3'UTR tend to participate in regulating mRNA stability or translation [5][6][7] . As an emerging theme, many RBPs are intimately involved in transcriptional regulation by interacting with promoter and enhancer noncoding RNAs [7][8][9] . Such position effects and regulatory rules highlight the value of developing transcriptome-wide methods for mapping the precise binding positions of a de ned RBP.
Methods currently available for pro ling RBP binding landscapes in living cells include CLIP-seq (crosslinking and immunoprecipitation sequencing, also known as HITS-CLIP), iCLIP, and eCLIP [10][11][12][13] . These three state-of-the-art approaches use a speci c antibody to pull down de ned RBP-associated RNA fragments from millions of cells and sequence them unbiasedly. Conceptually different from CLIP-based methods, an elegant technique called TRIBE fuses the catalytic domain of adenosine deaminases ADAR with individual RBP and deduces RBP targets by directly calculating the in vivo RNA editing sites 14 . Though TRIBE can map RBP targets from hundreds of cells, signi cant challenges arose from the low editing e ciency and duplex bias of the ADARcd domain 15 , which tends to cause frequent A-to-I editing at distal positions far from the bona de RBP binding sites. Moreover, ectopically expressed RBPs may perturb native RNA interaction networks. Therefore, mapping protein-RNA interactions in fewer cells under native conditions still represents a signi cant challenge.
To address this technical challenge, we have developed a LACE-seq method for unbiased mapping of precise RBP-binding positions in fewer cells or even single oocytes under native conditions. In brief, LACEseq includes the following steps: (1) cells are cross-linked with UV-C light, and the interested RBP-RNA complexes are immunoprecipitated from cell lysate and fragmented on the beads with micrococcal nuclease; (2) The 3' ends of fragmented RNAs are dephosphorylated and ligated with a 5' pre-adenylated linker containing four randomized nucleotides; (3) reverse transcription is performed on beads with a biotinylated primer containing the T7 promoter sequence; (4)   Please note that this is a modi ed protocol suitable for large amounts of cells, for which IVT is not necessary.

UV cross-linking of cells
1) Grow cells in 10 cm Petri dishes to reach 80% con uence, rinse twice with 5 ml of ice-cold 1 × PBS.
2) Place the dish on ice with the cover off, irradiate cells with UV-C light at 400 mJ two times.
3) Scrape the cells off from the dish into 5 ml of 1 × PBS, transfer cell suspension to a 50 ml centrifugation tube, and centrifugation at 500 g for 5 min at 4 °C.