GSM8005349: eCLIP-Seq, U2OS, input; Homo sapiens; OTHER
Protein-associated RNAs by eCLIP and input RNAs were extracted using acid phenol:chloroform:isoamyl alcohol (25:24:1, pH 6.6) (Invitrogen) and RNA cleanup was performed with RNA Clean & Concentrator-5 columns (Zymo). After reverse transcription, qPCR was performed to the Ct value of the cDNA with Power SYBR Green Master Mix (Applied Biosystems). eCLIP-seq libraries were prepared with primers containing Illumina adapter sequences using Q5 High-Fidelity 2× Master Mix (NEB) for 9 to 18 cycles and PCR products were purified with Agencourt AMPureXP beads (Beckman Coulter). After separating on a 3% Nusieve GTG agarose gel (Lonza), DNA ranging from 175 - 300 bp gel fragments was excised and extracted using the MinElute Gel Extraction Kit (Qiagen) and libraries were validated using the 1× High Sensitivity DNA Kit (Invitrogen).