title: GSM7256299: DDX3 WT Nuclear RNAseq replicate 1; Homo sapiens; RNA-Seq
library_construction_protocol: Approximately 5-10 million cells were washed and resuspended in PBS and 10% of cells were resuspended in RIPA buffer for the total fraction. The remaining 90% was pelleted and resuspended in 200ul cytoplasm lysis buffer (10mM Tris-HCl pH 7.0, 150mM NaCl, 0.15% NP-40) and incubated on ice for 5 minutes. Lysate was then layered on top of 450uL sucrose buffer (25% sucrose, 10mM Tris-HCl, 150mM NaCl) and centrifuged at 13,000 RPM at 4°C for 10 minutes to pellet nuclei. The top (cytoplasm) fraction was isolated. Nuclei were resuspended in 400uL nuclei wash buffer (1x PBS with 1mM EDTA, 0.1% Triton-X) and centrifuged at 3,500 RPM at 4°C for 1 minute. To isolate the nuclear fraction, the washed nuclei were resuspended in 125uL RIPA buffer, then to 100ul lysate 1ul of Proteinase K (Thermo Fisher cat: EO0491) was added, and incubated at 50C for 10 min. RNA was isolated from nuclear and cytoplasmic fractions with the Quick-RNA Microprep kit (Zymo cat: R1051). RNAseq libraries were prepared as per manufacturer's instructions using the NEBNext rRNA Depletion Kit V2 (NEB cat: E7400) and the NEBnext Ultra II Directional RNA Library Prep Kit (NEB cat: E7760).