title: GSM5471729: IPCtrl_2; Homo sapiens; RIP-Seq
library_construction_protocol: Cytoplasmic extracts isolated by hypotonic lysis in a Dounce homogenisator. Cells washed twice in MOPS-buffered saline. Cell pellet resuspended in 1,5 ml hypotonic lysis buffer per gram cells and lysis in a Dounce homogenisator. Cell debris removed by centrifugation and supernatant was used for Co-immunoprecipitation (IPP). For IPP 1 mg of cytoplasmic extract was diluted in 500 µl IPP buffer (20 mM HEPES, pH 7.4, 100 mM KCl, 5 mM magnesium acetate, 1 mM dithiothreitol, 0.025% Triton X-100, and protease inhibitors) and incubated with 30 µl of Protein G Sepharose™ 4 Fast Flow beads (GE Healthcare Life Sciences) coupled to a-hnRNP K antibody (Naarmann-de Vries et al. 2016) or a non-related control for 1.5 h at 4°C. Beads were washed twice with IPP buffer. Subsequent RNA isolation with TRIzol. 100 µg of cytoplasmic extract were used as an Input control for RNA analysis Quality checks of RNA samples were performed with the TapeStation 4200 using the Agilent RNA ScreenTape Assay kit. Quantification was performed using the Quantus RNA System (Promega). cDNA libraries were produced employing the TrueSeq Stranded Total RNA Library Preparation Kit with Ribo-Zero Gold Kit according to the manufacturer´s instructions (Illumina Inc., San Diego, CA, USA), using 500 ng input RNA or 10 µl for the immunoprecipitated samples, respectively. Quality and quantity of the cDNA libraries were assessed using the 4200 TapeStation (D1000 screen tape assay) and the Quantus dsDNA system, respectively.