title: GSM6456071: U2OS_iCLIP_GAR1_rep1; Homo sapiens; RNA-Seq
library_name: GSM6456071
library_construction_protocol: Protein-RNA complexes were fixed by exposing cells to 1500 J/m^2 254 nm UV light and collected by scraping. After nuclear fractionation, the lysate was treated with 0.4 Units RNaseI and 4 Units TurboDNase (both from Thermo Fisher Scientific) per 1ml of cell lysate at 1mg/ml protein concentration. Beads coupled with mouse IgG, dyskerin (Abnova, H00001736-m04) or GAR1 (Proteintech, #11711-1-AP) antibodies were used to precipitate protein-RNA complexes. RNA was ligated to a pre-adenylated infra-red labeled IRL3 adaptor. Complexes were resolved through SDS-PAGE and cut from the nitrocellulose membrane. RNA was then released from the membrane with proteinase K digestion and recovered by precipitation. RNA was retrotranscribed with Superscript IV Reverse Transcriptase (Thermo FIsher Scientific). The resulting cDNA was purified using AMPure XP beads (Beckman- Coulter), then circularised using CircLigase II (Epicentre), and purified again with AMPure XP beads. After PCR amplification, libraries were size-selected with Ampure XP beads and quality-controlled for sequencing.