GSM2177723: Control 293 cell; Homo sapiens; RNA-Seq
For samples 1-8, Cells (~50 millions) were harvested and lysed in 2ml RIPA buffer (50mM Tris-HCl pH 7.4, 1% NP-40, 0.5% sodium dexoycholate, 0.1% SDS, 1mM EDTA, 150mM NaCl, 1mM DTT, 50 units/ml RNase Out). The cell lysates were sonicated at Bioruptor (Diagenode) at â€˜high modeâ€™ for 60 cycles to shear to 400-800bp. After centrifuge, the clear lysate were immunoprecipitated at 4Â°C overnight. Beads were washed by low salt (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.1, 150mM NaCl), high salt (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl PH8.1, 500mM NaCl),  LiCl immune (0.25M LiCl, 1% NP-40, 1% deoxycholic acid, 1mM EDTA, 10mM Tris-HCl PH8.1), and TE buffer (1mM EDTA, 10mM Tris-HCl pH8.0). After proteinase K digestion and turbo DNase I (4U/sample) treatment, the RNA was recovered by acidic phenol/chloroform extraction and ethanol precipitation. For samples 9-10, total RNAs were extracted using the RNeasy kit (Qiagen). For mRNA-seq, 2 rounds of polyA+ selection was performed. For RIP-seq including the Input RNA-seq, no polyA+ or other selection was performed, followed by conversion to cDNAs. We used the stranded mRNA library generation kits per manufacturerâ€™s instructions (Agilent, Santa Clara, CA). Library construction consists of random fragmentation of the RNA, followed by cDNA production using random primers with inclusion of Actinomycin D in the first strand reaction. The ends of the cDNA are repaired, A-tailed and adaptors ligated for indexing (4 different barcodes per lane) during the sequencing runs. The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems, Woburn, MA) prior to cluster generation. Clusters were generated to yield approximately 725 K to 825 K clusters/mm2. Cluster density and quality were determined during the run after the first base addition parameters were assessed. We ran paired end 2x50bp sequencing runs to align the cDNA sequences to the reference genome.
GEO Accession
GSM2177723