Project description:To identify the directly bound transcripts of Flag antibody (the backgroud control for our METTL16 RIP-seq), RNA immunoprecipitation sequencing (RIP-seq) was conducted HEK293T. Briefly, HEK293T cells were infected with pmiRNA1-empty vector. Only the GFP-positive cells were used for study and expanded in DMEM medium.
Project description:We identified the mRNA targets of the insulin-like growth factor-2 (IGF2) mRNA-binding proteins 1, 2, and 3 (IGF2BP1/2/3) by RNA immunoprecipitation and sequencing (RIP-seq). HEK293T cells transfected with Flag-tagged IGF2BP1/2/3 plasmids were expanded and UV-crosslinked before harvest. We performed RIP of individual IGF2BP using anti-Flag antibody from nuclear extractions, and identified the associated mRNAs by next generation sequencing. More than 5000 transcripts, including protein coding and non-coding transcripts, were identified from each RIP-seq sample.
Project description:To identify the directly bound transcripts of METTL3, METTL14, and METTL16, RNA immunoprecipitation sequencing (RIP-seq) was conducted HEK293T stablely expressing METTL3, METTL14, and METTL16, respectively. Briefly, HEK293T cells were infected with lentivirus, pmiRNA1-3 x Flag-METTL3, pmiRNA1-3 x Flag-METTL14, and pmiRNA1-3 x Flag-METTL14, to overexpress the three METTL family members with 3 x Flag fused in the N-terminal. Only the GFP-positive cells were used for study and expanded in DMEM medium.
Project description:TDP43 and SRSF3 has been reported to be RNA-binding proteins; however their roles in breast cancer progression has not been examined previously. Here, we performed RNA-seq on MDA-MB231 cells stably expressed sh-control, shTDP43, shSRSF3 or sh-TDP43 and sh-SRSF3 using lentivirus in duplicates. In addition, MDA-MB231 cells with stable expression of flag-TDP43 or flag-SRSF3 were also generated by using lentivirus. RIP-seq was also applied to identify binding RNA against Flag antibodies.
Project description:In order to explore the mechanisms of Dmrt1 in regulating testicular immune homeostasis, we performed ChIP-seq analysis on primary SCs infected with a lentiviral vector expressing FLAG-tagged Dmrt1
