NextSeq 500 sequencing; RNA-Seq of purified P-bodies from HEK293 cells
RNA-Seq of purified P-bodies from HEK293 cells
P-bodies (cytosolic RNP granules) were purified as follow. GFP-LSM14A and GFP-LSM14A HEK293 cells were grown to 80% confluence in DMEM supplemented with 10% fetal calf serum. Starting from two plates of 15 mm for each cell line, cells were washed twice in PBS and scrapped in 2mL PBS. Cell pellets were frozen and stored at -80°C. Except stated otherwise, all the following steps were either conducted at 4°C or on ice. Pellets were suspended in lysis buffer (50 mM Tris (pH 7.4), 1 mM EDTA, 150 mM NaCl, 0,2% Triton X-100), in the presence of 65 U/mL RNaseOut recombinant ribonuclease inhibitor (Promega) and protease inhibitor cocktail without EDTA (Roche Diagnostics, Meylan, France). To accelerate cell lysis, cell extracts were passed 20-times through a syringe needle (27Gx3/4”, Nr. 20, 0.4X20mm, RW/LB, Henke Sass Wolf), for a total incubation time in lysis buffer of 20 min. Cells were spun for 5 minutes at 200xg to deplete nuclei. To eliminate remaining DNA contaminants, supernatants were supplemented with MgSO4 and CaCl2 (10 mM and 1 mM final, respectively) and were treated with 4 U/mL of RQ1 DNase (Promega) for 30 min at RT. Extracts were further spun for 7 min at 10000xg, and pellets were re-suspended into 40 L of lysis buffer with 80 Units of RNaseOut recombinant ribonuclease inhibitor (Promega). This organelle enriched 10000xg pellet fraction was named the pre-sorted fraction. Next, P-bodies were sorted on a cell sorter (MoFlo Astrios EQ, Beckman Coulter) equipped with a nozzle size of 70 m, and working at 66 000Hz with a 30psi pressure. Particles were detected according to their Forward-scattered light (FSC) and through their green fluorescence using the 488nm excitation laser and the 526/52 band pass filter. To define the P-body sorting window, we compared an extract in which P-bodies were fluorescently labelled (GFP-LSM14A) to an extract in which P-bodies were not fluorescent (GFP-LSM14A). The purity mode (1-2 envelop) and a differential pressure of 0.5 were applied for sorting. Note that the starting dilution of the pre-sorted extract was critical for the purity of sorted P-bodies. To this end, a dilution of the presorted extract (10 l in 2 mL lysis buffer) was adjusted to detect 10000 Event Per Second (EPS). P-Bodies represented 10% of all detected EPS, and 1000 P-bodies were sorted per second for a sorting efficiency of 85%. After 6 hours of sorting, we cumulated 21E6 sorted particles. The collected fraction was spun for 7 min at 10000xg to pellet the sorted P-bodies. The sorted P-bodies and matching pre-sorted fractions were stored at minus 80°C. To fluorescently labels P-bodies, Human embryonic kidney HEK293 were stably transfected with a GFP-LSM14A plasmid. Briefly, HEK293 cells were maintained in DMEM supplemented with 10% fetal calf serum. Plasmid transfections were performed with 1 µg DNA per 35 mm diameter dish using GenJet Plus DNA (SignaGen Laboratories, Rockville, USA ) according to the manufacturer's protocol. The GFP-LSM14A plasmid was generated by cloning in frame the human LSM14A isoform1 (NCBI Reference Sequence: NM-001114093.1) ORF fragment (1392 bp) into pEGFP-C3 vector (BamHI/EcoR1 restriction sites), using In-Fusion HD cloning kit (Takara Bio, USA). For stable transfection, HEK293 cells were transfected with GFP-LSM14A plasmid as described above. 48 h later, cells were split in 100 mm diameter dishes and selected in the presence of 50 μg/ml geneticin sulfate (Invitrogen, France). After 3 days of selection, cells were further split to clonal dilution in 96-well plates. Clones of interest were selected using fluorescence microscopy to control for GFP-LSM14A expression levels and subcellular localization. Following Trizol LS (Invitrogen) extraction, the P-body sorted fractions gave a yield of ~30 ng of RNAs. Starting from 3 biological replicates, cDNAs were synthetized using random priming for the sorted P-bodies and pre-sorted fraction RNA libraries (Ovation RNA-Seq system NuGEN)
Experimental Factor: pre-sorted fraction
fraction