GSM6696309: HAP1 MATR3 KO MATR3 PAR-CLIP; Homo sapiens; RIP-Seq
UV-crosslinked cells were scraped off the dishes, lysed, Rnase T1 treated, and then immunoprecipitated using anti-FLAG magnetic beads. Beads were washed and resuspended in immunoprecipitation wash buffer. RNA residing in the immprecipitate was further trimmed with RNase T1, washed three more times in high-salt lysis buffer, and finally resuspended in one bead volume of dephosphorylation buffer. (see Methods section for exact amounts, concentrations and timings used) 3' ends of RNA fragments were dephosphorylated in calf intestinal phosphatase and RNA was radioactively 5' end-labeled using T4 polynucleotide kinase and P-32-gamma-ATP. Protein-RNA complexes were separated by SDS-PAGE, transferred to nitrocellulose and visualized with autoradiography. Radioactive bands corresponding to the MATR3 rbionucleoprotein complex were recovered, and protein was removed via proteinase K digestion. RNA was then isolated by acidic phenol/chloroform extraction and ethanol precipitation. The recovered RNA was converted into a cDNA library using a standard small RNA library protocol (Hafner et al., Methods 2008) and sequenced on an Illumina NovaSeq 6000 instrument. (see Methods section for exact amounts, concentrations and timings used)