GSM7718986: TIS11B_iKO_cyto_rep1; Homo sapiens; RNA-Seq
20 hours after transfection cells were rinsed once with ice cold PBS then scraped in 10 mL ice cold PBS and pelleted at 300g. Pellets from two 10 cm plates were resuspended in 1 mL ice cold hypotonic isolation buffer (225 mM mannitol, 75 mM sucrose, 20 mM Tris-HCl pH 7.4, 0.1 mM EDTA). Cells were lysed with 50 strokes in a 1 mL dounce homogenizer with pestle on ice in order to shear the nuclei from the cytoplasm. Nuclei were pelleted with a two-minute spin at 600g. Subcellular organelles were then pelleted from the supernatant with a 15-minute spin at 7000g and resuspended in ice cold PBS for fluorescence activated particle sorting. Particles were sorted on a BD FACSAria III cell sorter equipped with 70 um nozzle size. mCherry-TIS11B and GFP-SEC61B particles were first detected by fluorescence using the 594 nm and 488 nm excitation lasers respectively, and 405 nm excitation laser for DAPI. A particle gate was drawn to include mCherry-positive or GFP-positive events and DAPI-negative events in order to exclude any remaining nuclei. Particles were then selected for sorting by size and granularity using forward-scattered light (FSC) and side-scattered light (SSC) respectively. The FSC threshold was decreased from 5,000 to 800 in order to visualize subcellular particles. Sorting was performed on purity mode with an average of sorting speed of 150 particles/second. Particles were sorted directly into 1 mL of TRIzol solution in Eppenforf tubes, holding 180,000 particles per tube. RNA-extraction was then performed for each tube separately. Total RNA yield was combined before for library prep. For each replicate about 1.5 million TIS11B granule particles were collected and 2.0 million ER particles. For cytosolic extraction, 20 hours post transfection cells were rinsed once in the dish with ice cold PBS. After aspirating PBS, 400 ul ice cold digitonin solution (40 ug/mL digitonin, 150 mM NaCl, 20 mM HEPES pH 7.4, 0.2 mM EDTA, 2 mM DTT, 2 mM MgCl2) was added and incubated on a shaker at four degrees for ten minutes. After incubation, the digitonin solution now containing the cytosolic extract was pipetted from the plate and spun at 20,000g for one minute to pellet any floating cells. 200 ul of cytosolic extract was added to 1 mL TRIzol solution for RNA extraction. Library preparation and Illumina sequencing were performed by the MSKCC Integrated Genomics Operations core facility. mRNA libraries were prepared by poly-A selection.The libraries were multiplexed and after paired-end 50 bp sequencing 10-20 million reads per sample were acquired.