Efficient and sensitive profiling of RNA-protein interactions using TLC-CLIP [dataset 2]
RNA-binding proteins are instrumental for post-transcriptional gene regulation, yet transcriptome-wide methods to profile RNA-protein interactions remain technically challenging. We present an improved library preparation strategy for cross-linking and immunoprecipitation (CLIP) that involves tailing and ligation of cDNA molecules (TLC) for increased sensitivity and efficiency. TLC-CLIP eliminates time-consuming purifications, reduces sample loss, and minimises experimental steps, allowing precise profiling of RNA-protein interactions from limited starting material at nucleotide resolution. Overall design: We have generated benchmarking libraries for 4 well-studied RNA-binding proteins from 50.000 293T cells in duplicates, including additional control samples comparing different RNase concentrations, additional adapter removal, omitting PAGE purification and profiling of co-purifying fragments from non-crosslinked samples. We further demonstrate the high sensitivity of our protocol by profiling hnRNPc and RBFOX2 from only 10,000 and 1,000 cells. To integrate our CLIP data with functionally relevant sites we have performed RNA-Seq in 293T knockout cells for RBFOX2 to identify alternatively spliced exons.