GSM5823381: iCLIP_endogenous_PURA_NPC_s4; Homo sapiens; RIP-Seq
Immunoprecipitation of PURA from cell lysates was performed using anti-PURA12D11-coated Dynabeads protein G (Thermo Fisher Scientific). Subsequently, co-purified RNAs were dephosphorylated at the 3' end, the 3' adapter was ligated and the 5' end was labeled with radioactive isotopes (γ-32P ATP; Hartmann Analytic). Samples were loaded onto a NuPAGE 4-12% Bis-Tris pre-cast SDS gradient gel (Thermo Fisher Scientific), transferred to a nitrocellulose membrane (GE Healthcare) and visualized using a Phosphoimager (Fujifilm). After cutting the correct region from the nitrocellulose membrane using a cutting mask, RNA was isolated by Phenol/Chloroform/Isoamylalkohol (pH=8.0, Sigma Aldrich) and the mix was transferred to a 2 ml Phase Lock Gel Heavy tube (Quantabio). After separation of the phases the RNA was precipitated by the addition of 0.75 μl GlycoBlueTM Coprecipitant (ThermoFisher), 40 μl 3 M sodium acetate pH 5.5 and 1 ml 100 % Ethanol. It was mixed and stored at -20 °C for at least two hours. Reverse transcription was performed on the resulting purified RNA using Superscript III (Thermo Fisher Scientific) according to manufacturer's instructions. Then cDNA was immobilized on MyONE SILANE beads (Thermo Fisher Scientific) as described by the manufacturer and the second adapter was ligated. The library was amplified for six PCR cycles with short primers followed by a ProNex size selection step (Promega) in a 1:2.95 (v/v) sample/bead ratio according to manufacturer's instructions. The size-selected library was amplified for 22 cycles. Afterwards, a second size selection was performed using ProNex chemistry (Promega) at 1/2.4 (v/v) sample/bead ratio according to manufacturer's instructions and cDNA was eluted in 20 μl MQ water. The final library was analyzed using a D1000 Chip (Agilent) in a Bioanalyzer (Agilent). Samples were equimolarly mixed before sequencing on an Illumina HiSeq 1500 platform with 75-nt single-end reads and 6.7-7.9 million reads per sample