U2 IP-seq from K562 cells expressing SF3B1-3xFlag WT or SF3B1-3xFlag K700E
The branch site / 3' splice site recognition machinery is a hotspot for disease-associated mutations. A single amino acid substitution K700E in the U2 snRNP-specific protein SF3B1 is frequently associated with cancer and is particularly common among myelodysplastic syndromes. The goal of this study is to employ the U2 IP-seq strategy in examining the differences in branch site selection in cells expressing wild type SF3B1 vs those carrying the K700E mutation. Overall design: K562 erythroleukemia cell line was engineered in Manley lab to express a 3x-Flag tagged version of SF3B1, either carrying the K700E mutation or retaining the original sequence (Zhang J et al 2019, PMID: 31474574). These cells were grown under standard conditions (in IMDM medium supplemented with 10% FBS), harvested and washed with 1x PBS. High molecular weight nuclear extracts were prepared as previously described (Damianov A et al 2024, PMID: 38537639). Precatalytic complexes carrying branch site sequences were affinity purified either with anti-SF3A2 antibody, each in a triplicate, or with anti-FLAG antibody recognizing the epitope tagged SF3B1. In parallel controls were carried out without antibody, or using an aliquot of the extract without affinity purification. After deproteinization, the U2 snRNA fragments were selectively degraded with antisense DNA oligonucleotide and RNase H. Sequencing libraries carrying inline barcodes were constructed using the iCLIP library reagents as in previous IP-seq experiments (Damianov A et al 2024, PMID: 38537639). and were sequenced in two S4 2x 100 bp lanes. After demultiplexing and removal of PCR duplicates, clusters of mapped reads will be determined. Branch site clusters were annotated separately as in the IP-seq analyses from 293Flp-In cells expressing Flag-tagged proteins. Branch point analysis were also performed as before.