GSM8654612: Panc-1 negative control Rep B IgG; Homo sapiens; RIP-Seq
30 x 10^6 Panc-1 cells, treated or not for 16 h with 1 µM PDS, were collected and then lysed for 10 min. with Hypotonic Lysis buffer (5 mM Pipes, 85 mM KCl, 0.5 % NP40) containing protease inhibitor cocktail. Pellets containing nuclei were discarded while cytoplasmic fraction was diluted 1:1 in G4RP buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT and 0.5 % NP40 in nuclease-free water). After preclearing, lysates were incubated with  3 μg of BG4 (Merck) antibody and 8 μl Dynabeads Protein G for 6h at 4°C. After washings, beads were incubated with 1ml of Tri-Reagent (Sigma-Aldrich, Germany) to extract RNA with a phenol/chloroform extraction method. RNA was DNAseI (Thermo Fisher Scientific) treated to digest genomic DNA. Concentration, integrity and purity of RNA were checked using Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit). 10 ng of RNA was digested with DNAseI (NEB), freed from rRNA (using the ribo-zero method) and reverse transcribed to obtain cDNA. Adapters were ligated to the fragmented cDNA after end repair and A-tailing according to  Illumina protocols. 16 rounds of PCR amplification were performed to enrich the cDNA fragments. The PCR products were then purified using Ampure XP beads (Agencourt) and subjected to paired-end 100 sequencing at the Genewiz Genomics facility (Genewiz, Germany).