GSM4861175: nuclear_vcp_rep1; Homo sapiens; RNA-Seq
Subcellular fractionation was achieved using the Ambion PARIS kit (ThermoFisher Scientific). The cytosolic fraction was obtained by lysing cells in ice-cold cell fractionation buffer for 5 minutes, disrupting plasma membranes whilst leaving nuclear membranes intact. Lysates were centrifuged for 3 minutes at 500 x g at 4°C. Supernatant was further centrifuged at maximum speed at 4°C for 1 minute, and the resulting supernatant was processed as the cytosolic fraction. Nuclear pellets from the first centrifugation step were gently washed with cell fractionation buffer and then lysed on ice for 30 minutes in 8M Urea Nuclear Lysis Buffer. The resulting nuclear fraction was homogenised using a QIAshredder (Qiagen) to shred chromatin and reduce viscosity. Both lysis buffers were supplemented with 0.1 U/μl RiboLock RNase Inhibitor (ThermoFisher Scientific) and HALT Protease Inhibitor Complex (ThermoFisher Scientific). Poly(A)+ selected reverse stranded RNAseq libraries were prepared from whole astrocyte and their nuclear and cytoplasmic fractions obtained from astrocyte differentiation from two control and two VCP mutant cell lines using the KAPA mRNA HyperPrep Library kit for Illumina®, with 50ng of total RNA as input.