GSM7157903: 4 TSC Total RNA; Mus musculus; RNA-Seq
Primary NSC cultures were passaged once prior to isolations. 100 mg/ml (66.6uL) cycloheximide (CHX, VWR, #94271) was added to each dish and incubated at 37°C for 15 minutes. The cells were dissociated in 500 mL lysis buffer (0.25 M sucrose, 50 mM Tris/HCl (pH7.5, Sigma Aldrich, #93363), 5 mM MgCl2 (Sigma Aldrich, #M1028), 25 mM KCl (Sigma Aldrich, #P9333), 200 µg/ml CHX (VWR, #94271), 1X HALT protease inhibitor (Thermo Scientific, #87785), 1 mM dithiothreitol (DTT, Enzo, #ALX-280-001-G005), 100 U/ml recombinant RNAsin ribonuclease inhibitor (Promega, #N2511), scraped into a 1.5 mL microcentrifuge tube, and incubated for 15 minutes on ice. Lysates were homogenized five times with a Dounce homogenizer. 50 mL was removed and 150 mL TRIzol LS reagent (Ambion, #10296010) was added for RNA Total isolation. The remaining lysates were centrifuged for 10 minutes at 500 x g at 4°C. 10% sodium deoxycholate and 10% NP-40 was added, and the samples were incubated on ice for thirty minutes. Prepared lysates were loaded onto 10 mL continuous sucrose gradients that consisted of 17.5-50% sucrose (as well as 30 mM Tris-HCl (pH7.5, Sigma Aldrich, #93363), 30 mM MgCl2 (Sigma Aldrich, #M1028), 600 mM NaCl (Fisher Scientific, #BP358-10), 200 µg/ml CHX (VWR, #94271), 2 mM DTT (Enzo, #ALX-280-001-G005), and UltraPure DEPC treated H2O (Invitrogen, #750023). The gradients were balanced using lysis buffer and were centrifuged for 1.5 hours at 280,000 x g in a SW-41 Ti swinging bucket rotor with the acceleration speed set to max and the deceleration speed set to coast. Seven 1080 mL fractions were then removed from the top of the sucrose gradients leaving 3 mL of the heaviest sucrose fractions. TRIzol LS reagent (9 mL) (Ambion, #10296010) was added and stored at -80°C overnight. The solutions were then split into two equal parts and 1.2 mL of chloroform (Sigma Aldrich, #372978) was added to each tube. The tubes were incubated for three minutes before centrifugation at 12,000 x g for 15 minutes at 4°C. The aqueous layer was then removed and 3 mL isopropanol (Fisher Chemical, #A416-1) and 1 mL GlycoBlue Blue coprecipitant (Invitrogen, #AM9516) was added. The tubes were then moved to the -80°C freezer to precipitate overnight. Samples were then centrifuged for ten minutes at 12,000 x g at 4°C and the supernatants discarded. Pellets were resuspended in 6 mL 75% EtOH (Sigma Aldrich, #E7023) and vortexed before centrifugation for five minutes at 7,500 x g at 4°C. The supernatants were discarded, and the pellets allowed to air dry for 8 minutes before resuspension in 50 mL UltraPure Distilled water (Invitrogen, #10977-015). The samples were incubated for 15 minutes at 57°C in a water bath before being recombined into one polysomal RNA fraction each. Chloroform (Sigma Aldrich, #372978) was added to each cell lysate that was collected during polysomal profiling as a total RNA fraction. The tubes were incubated for three minutes before centrifugation at 12,000 x g for 15 minutes at 4C. The aqueous layer was then removed and 100 µL isopropanol (Fisher Chemical, #A416-1) and 1 mL GlycoBlue Blue co-precipitant (Invitrogen, #AM9516) was added. The tubes were then moved to the -80°C freezer to precipitate overnight. Samples were centrifuged for ten minutes at 12,000 x g at 4°C and the supernatants discarded. Pellets were resuspended in 200 µL 75% EtOH (Sigma Aldrich, #E7023) and vortexed before centrifugation for five minutes at 7,500 x g at 4°C. The supernatants were discarded, and the pellets allowed to air dry for 8 minutes before resuspension in 50 mL UltraPure Distilled water (Invitrogen, #10977-015). The samples were incubated for 15 minutes at 57°C in a water bath before library prep. Libraries were constructed using the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (New England Biolabs Inc., #E7770S) and the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs Inc., #E7490S). These libraries were dual-indexed using the NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1) (New England Biolabs Inc., #E7600S). The libraries were quantified and qualified using Qubit (Thermo-Fischer, #Q32852) and TapeStation (Agilent, # 5067-5579).