GSM5269437: PROseq_Lymphblastoid cells_D21_37C_rep1; Homo sapiens; OTHER
Nuclei isolations: Cells were grown in suspension in T75 [MAA1] flasks. After cell treatments, 10 ml aliquots of cells in media were washed 3X with cold PBS (phosphate buffer saline). Cells were next incubated on ice with buffer 1 containing 10 mM Tris pH 7.5, 2mM MgCl2, and 3 mM CaCl2 in DEPC (1% diethylpyrocarbonate treated) water, before centrifuging for 10 minutes at 1000 x G at 4°C. Buffer was then aspirated and cells were resuspended in 1ml of buffer 2 containing 10 mM Tris pH 7.5, 2mM MgCl2, and 3 mM CaCl2, 0.5% IGEPAL (octylphenoxy poly(ethyleneoxy)ethanol, branched, a nontoxic NP40 alternative), 10% glycerol, 2U/ml of SUPERase-IN, 1X protease inhibitor cocktail, and 1mM DTT (dithiothreitol) in DEPC treated water. After resuspension, 9 ml more of buffer 2 was added, samples were mixed by inverting the tube, and samples were then centrifuged for 10 minutes at 1000xG at 4°C. Solution was then aspirated, leaving behind cell nuclei. Nuclei were cleaned up by repeating the process of resuspending in 1ml of buffer 2, bringing solution up to 10 ml total buffer 2, spinning samples at 1000xG for 1 minutes, and aspirating media. White nuclei pellets were then resuspended in 1ml of buffer 3 containing 50 mM Tris pH 8.3, 5 mM MgCl2, 40% glycerol, and 0.1 mM EDTA (ethylenediaminetetraacetic acid) pH 8.0 in DEPC treated water. Nuclei in buffer 3 were transferred to pre-lubricated 1.7ml tubes, centrifuged for 5 minutes at 600xG at 4°C, and aspirated. Nuclei pellets were then resuspended in 500 ul of buffer 3, the centrifugation process was repeated, and final nuclei pellets were suspended in 110 ul buffer 3. A 1 ul aliquot of nuclei suspensions was diluted in PBS for nuclei quantification. Samples were diluted to make 100ul aliquots of 10 million nuclei each, and then flash frozen in liquid nitrogen and stored at -70°C. Precision run on sequencing experiments (PRO-seq) were carried out according to the PRO-seq protocol (Mahat et al). ~10 million nuclei were used per sample. Prior to the run on experiment, Drosophila S2 cell line nuclei were spiked into each sample at approximately 1% for potential use in normalizing samples. Run on buffer containing 25 uM biotinylated rCTP, 25 uM rCTP, and 125 mM rATP, rGTP, and rUTP was used. Ampure bead cleanups were conducted at 1:1 ratios on the samples before and after the final PCR cleanup step.