GSM4631213: CTRL4_35_Cytoplasmic_1; Homo sapiens; RNA-Seq
Biochemical subcellular fractionation was achieved for all cell stages using the Ambion PARIS kit (ThermoFisher Scientific) cell fractionation buffer, following the manufacturer's general protocol, and an 8M Urea Nuclear Lysis Buffer prepared in house. Initially, cells were washed once using ice-cold PBS. Cytosolic fraction was then obtained by lysing cells directly in ice-cold cell fractionation buffer (ThermoFisher Scientific) for 3-15 minutes, thus disrupting plasma membranes, whilst leaving nuclear membranes intact. Lysates were then centrifuged for 3 minutes at 500 x g at 4C. The supernatant was then collected, further centrifuged at maximum speed in a bench centrifuge at 4C for 1 minute, and the resulting supernatant was then processed as cytosolic fraction. Nuclear pellets from the first centrifugation step were gently washed once with cell fractionation buffer and then lysed on ice for 30 minutes in 8M Urea Nuclear Lysis Buffer, containing 50mM Tris-HCL (pH 8), 100mM NaCl, 0.1% SDS, and 1mM DTT. The resulting nuclear fraction was then homogenised using a QIAshredder (QIAGEN) to shred chromatin and reduce viscosity, before being further processed for RNA extraction.  Both lysis buffers were supplemented with 0.1 U/µl RiboLock RNase Inhibitor (ThermoFisher Scientific) and HALT Protease Inhibitor Complex (ThermoFisher Scientific). Total RNA was extracted from cells using the Maxwell RSC simplyRNA cells kit (Promega), that includes DNase treatment, according to manufacturer's instructions. 500ng total RNA input was used with the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®, with NEBNext® Poly(A) mRNA Magnetic Isolation Module.
GEO Accession
GSM4631213