|Public on Jul 02, 2021
|cell line: MCF-7
antibody: YTHDF2 (Cat#ARP67917_P050)
cell culture: DMEM + 10% FBS
|20M cells were UV-crosslinked, lysed and sonicated. RNA was fragmented and protein-RNA complexes were immunoprecipitated with a YTHDF2 antibody. Inputs (2% of lysate) were saved and run alongside IP samples. IP samples were stringently washed, and for all samples the RNA was dephosphorylated, followed by on-bead ligation of barcoded RNA adapters to the 3’ end. RNA-protein complexes were run on standard protein gels and transferred to nitrocellulose membranes where the RNA in the region 65 kDa – 140kDa was excised off the membrane and proteinase K treated. RNA was then reverse transcribed followed by treatment with ExoSAP-IT to remove excess oligonucleotides. Samples were cleaned up with Dynabeads MyOne Silane and subject to qPCR to determine the appropriate number of PCR cycles.
|Illumina HiSeq 4000
|Library strategy: eCLIP-seq
Raw sequencing reads were trimmed for adapter sequences and barcode sequences using cutadapt. Trimmed reads were mapped against RepBase to remove reads mapping to repetitive sequences. Remaining reads were mapped to the appropriate genome build using STAR aligner. Read densities were calculated to identify eCLIP peaks.
Supplementary_files_format_and_content: input normalized and filtered peaks in BED format and Peaks files (bigwigs).
|Sep 11, 2019
|Last update date
|Jul 03, 2021
|2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab
|Inhibition of YTHDF2 triggers proteotoxic cell death in MYC-driven breast cancer