title: RNA-seq of purified p-bodies or their associated cytoplasm from different cell cycle stages in HEK293 cells
design: RNA-seq of purified p-bodies or their associated cytoplasm from different cell cycle stages in HEK293 cells
library_construction_protocol: Cells expressing LSM14A-GFP-labeled PBs (synchronized or not) were allowed to grow to ~70-80% confluency in 15cm plates. Cells were washed twice with PBs to remove medium and scrapped off and collected in 2ml Eppendorf tubes. Cells were then centrifuged at 500g for 5 mins and the supernatant was discarded. Pellets were flash frozen with liquid nitrogen and stored at -80°C until 1 day before sorting. Cells were then lysed on ice with a buffer containing 50mM Tris pH 8, 1mM EDTA, 150mM NaCl, and 0.2% triton in distilled water supplemented with 65U/ml RNase out (Promega) and 2x protease inhibitor cocktail (Roche). Each cell pellet was suspended in 1.5ml lysis buffer, mixed by pipetting a few times, and kept on ice for 20 mins. Then, samples were centrifuged for 5 mins at 500g to pellet nuclei. The supernatant containing cytoplasmic organelles, including PBs, was then transferred to a new tube taking care not to disturb the nuclei. The supernatant was then supplemented with 10mM MgSo4, 1mM CaCl2, and 4 U/ml RQ1 DNase (Promega) and incubated at room temperature (25°C) for 30 mins while avoiding direct sources of sunlight. Next, the sample was centrifuged at 10,000g for 7 mins at 4°C. The supernatant was then gently aspirated taking care not to disturb the organelle-rich pellet. This pellet was then resuspended in 30μl lysis buffer supplemented with 40U RNase out. This corresponded to the pre-sorted cytoplasmic fraction. 3 independent pre-sorted fraction preparations for each cell cycle phase were kept aside for RNA-seq analyses to compare with their sorted counterparts. The pre-sorted fraction was stored at -80°C and prepared the night before sorting.After sorting, purified PBs were collected and centrifuged at 10,000g for 7 mins in 2ml Eppendorf tubes to concentrate the sample (taking great care not to disrupt sorted PBs when discarding the supernatant). ~6 hours of active sorting were done per day and 2-3 days of sorting output were combined together to constitute a single replicate for RNA-seq for each cell cycle phase. Purified PBs were stored at -80°C until the purification pipeline was complete. HEK293 cells expressing GFP-LSM14A were grown in Dulbecco's modified Eagle's Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich),100 U/mL penicillin/streptomycin (Sigma-Aldrich), and selected under 500 μg/ml G418 (Gibco). RNA from each fraction was extracted using trizol. A total of 5-10ng RNA per replicate per condition was obtained for the sorted p-body fraction.  cDNAs were synthesized using random priming. 2 ng of total RNA were amplified and converted to cDNA using NuGEN's Ovation RNA-Seq kit.