title: GSM5472014: Doxo-Nuc-HuR-n2; Homo sapiens; RIP-Seq
library_construction_protocol: For preparation of cytosolic and nuclear extracts, cells were washed twice with ice-cold phosphate buffer saline (PBS) and harvested by scraping. Cells from each plate were pelleted in microfuge tubes, resuspended in 200 μL lysis buffer (10 mM Tris-HCl pH 8,140 mM NaCl, 1.5 mM MgCl2, 10 mM EDTA, 0.5% NP40 and protease inhibitor cocktail) and incubated on ice for 5 min followed by centrifugation at 470 g for 5 min at 4 °C. The supernatant was stored as cytosolic fraction and the pellet was washed twice gently with lysis buffer before being resuspended in lysis buffer as nuclear fraction. For total cell lysates, cells were lysed directly by pipetting in lysis buffer. The cell lysate fractions were then sonicated (12 cycles, 30:30) on a Diagenode sonicator. CLIP-seq procedure (iCLIP): Individual-nucleotide resolution Cross-Linking and Immuno Precipitation (iCLIP) experiments were performed as described (Huppertz et al 2014) with minor modifictaions, as detailed below, using protein lysates (whole cell/ nuclear/cytosolic) from MCF-7 cells treated or not with Doxo for 6 hours (2 replicates per condition). Briefly, 85-90% confluent MCF-7 cells in 10 cm dishes were irradiated with UV- light (254 nM) at 400 mJ/cm2 to crosslink protein-RNA interactions using Stratalinker 1800 (Stratagene). After washing cells with ice-cold PBS, cell fractions were obtained as described above. The lysates were sonicated, incubated with 800 µl RIPA buffer for 30 min. 10 µl RNase 1 (1:250) was added along with 2 µl of turbo DNAse and incubated at 37˚C for 3 min at 1,100 rpm in a table-top Thermomixer (ThermoFischer Scientific) followed by incubation on ice for 3 min. RNAse inhibitor (ThermoFischer Scientific) was added and the lysates were centrifuged at 22,000 rcf for 10 min at 4˚C to remove debris. Immunoprecipitation of HuR-RNA complexes was performed using 5 μg of anti-HuR mAb coupled to protein G dynabeads (ThermoFischer Scientific). IP with anti-mouse IgG (Santa Cruz Biotechnologies) was used as negative control. After washing twice with high-salt buffer RIPA-S (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS and 0.5% sodium deoxycholate) and once with PNK washing buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% Tween-20), one tenth of the sample were labelled with 32P-ATP using PNK, whereas an RNA Linker was ligated to the rest of the sample after RNA de-phosphorylation. RNA-protein complexes were separated by electrophoresis (SDS-Page) and transferred to nitrocellulose membranes. HuR RNA complexes higher than 55 KDa were marked after visualization by autoradiography. RNA extraction was performed by incubating the nitrocellulose fragment at 37°C for 10 min with proteinase K in PK buffer (100 mM This-Cl pH 7.5, 50 mM NaCl and 10 mM EDTA). 200 μl of PK buffer containing urea (7 M) was added to each sample and further incubated at 37°C for 20 minutes. RNA was isolated by acid-phenol/chloroform extraction and ethanol precipitation. RNA was reverse transcribed into cDNA using RCLIP primers and SuperScript III reverse transcriptase. After cDNA purification using 6% TBE-urea gels, cDNA was circularized and amplified by PCR using Solexa P5/P7 primers (Illumina). After cDNA purification using 6% TBE-urea gels, cDNA was circularized and amplified by PCR using Solexa P5/P7 primers (Illumina). cDNA libraries from independent experiments were prepared, purified using Agencourt AMPure XP (Beckman Coulter) magnetic beads, quantified with Quant-iT Picogreen dsDNA kit (ThermoFisher Scientific), and run on Experion automated electrophoresis system (BIO-RAD). Pooled libraries were quantitated by qPCR (KAPA Library Quantification Kits Illumina Platforms, Roche), diluted to 12 pM, and subjected to single-end, 50 bp sequencing using the HiSeq 2500 machine (Illumina).