title: GSM5365952: C9orf78 rep2; Homo sapiens; OTHER
library_construction_protocol: Cells were solubilized with 1X PBS with additional 0.3M NaCl 1% Triton™ X-100 and 0.2% TWEEN® 20 Flp-In™ T-REx™ 293 cells expressing either 3xFLAG-BIO tagged C9ORF78 or 3xFLAG-BIO tagged GFP were induced with 0.1µg/mL doxycycline for ~16hrs and UV-crosslinked with 0.2mJ/cm2 UV-C light. Target proteins were purified using MyONE C1 streptavidin beads, after a partial RNase digestion using RNAse1 and end-repair with T4 PNK, uniquely barcoded s-oligos were ligated to each sample (2 biological replicates). After several stringent washes (up to 1% SDS), biological replicates are merged, and bead-bound RNA was released with proteinase K and purified with Oligo Clean and Concentrator columns. RNA was then reverse-transcribed and treated with RNaseH to phosphorylate 5'-ends of cDNA. The cDNA was then circularized with CircLigaseII, amplified with Q5 polymerase and sequenced on an Illumina platform (100bp, PE sequencing). FLASH (Ilik et al., 2020)