title: GSM8656793: U2OS cells, untreated, whole cell lysate, replicate 1; Homo sapiens; RNA-Seq
library_construction_protocol: At assay endpoint cells were trypsinized, counted using a hemacytometer, pelleted, washed with PBS, and moved immediately to cellular fractionation. Cells were lysed in a hypotonic lysis buffer and left to swell on ice for 10 minutes. 1/3 of the lysate was removed and saved as the whole cell lysate (WCL) fraction. The samples were then spun for 30 seconds at 12000xg at 4 degrees. Supernatant was saved as the cytoplasmic (CYT) fraction. nuclei were washed 1x in wash buffer, the lysed in nuclei lysis buffer. Lysates were all normalized for differences in surfactant concentrations using a 10x nuclear lysis buffer. Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, 74134) according to manufacturer protocol. To facilitate absolute RNA quantification, polyadenylated ERCC standards (Thermo Fisher Scientific, 4456740) were diluted 1:10 in nuclease-free water, and added to each sample at 1 µL/1,000,000 cells (or equivalently, 1,000,000 cells worth of nuclei or cytoplasm). ERCC spike-ins were added immediately following lysis of cells in RLT-plus buffer, and prior to all subsequent total RNA extraction steps using the RNeasy Plus kit. mRNA was isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, E7490). RNAseq libraries were prepared with the NEBNext Ultra II Directional RNA Library Prep Kit using the NEBNext Multiplex Oligos for Illumina (New England Biolabs, E7765 and E7600).