Therefore, two types of cDNAs are generated: truncated cDNAs (which never contain the 5’-marker) and read-through cDNAs (some of which contain the 5’-marker). The peptide that remains at the crosslink site impairs reverse transcription and commonly leads to truncation of cDNAs at the crosslink site. Reverse transcription is performed with a primer that includes a barcode (orange) containing both an experimental identifier and a unique molecular identifier (UMI) (step 6). After SDS-PAGE purification (step 4), the crosslinked RBP is removed through proteinase K digestion and purification of RNA fragments since the ligation reaction is not 100% efficient, only a subset of the fragments contains both the 3’-adapter and the 5’-marker (step 5). Ligation of a 3’-adapter (step 3a) is followed by ligation of a 5’-marker that is unique to the modified protocol (red balloon, step 3b). After lysis, the crosslinked RNA is fragmented by a limited concentration of RNase I, and the RNA fragments are then co-immunoprecipitated with the RBP (step 2). Before lysis, cells or tissues are irradiated with ultraviolet (UV) light, which creates a covalent bond between proteins and RNAs that are in direct contact (step 1). Thus, cDNA start sites confidently identify a protein's RNA-crosslink sites and we can account for the impact of read-through cDNAs by commonly adding a 5'-marker.Įukaryotic initiation factor 4A-III (eIF4A3) High-throughput sequencing Polypyrimidine tract binding protein 1 (PTBP1) Protein–RNA interactions iCLIP.Ī) A schematic description of the modified iCLIP protocol. This finding in turn indicates that most of the reads in our iCLIP libraries are a product of truncation with valuable information regarding the proteins' RNA-binding sites. By applying this modified iCLIP protocol to PTBP1 and eIF4A3, we found that the start sites of read-through cDNAs are enriched in adenosines, while the remaining cDNAs have a markedly different sequence content at their starts, preferentially containing thymidines. To this end, we added an oligonucleotide to the 5'-end of RNA fragments-a 5'-marker-to mark the read-through cDNAs. A large proportion of these cDNAs would be undesirable in an iCLIP library, as it could affect the resolution of the method. We established a modified iCLIP protocol, called 'read-through marking', which facilitates the detection of cDNAs that have not been truncated upon encountering the RNA-peptide complex during reverse transcription (read-through cDNAs).
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