GSM7036360: Total_Chtop_mEx5_RESC_BR4: N2a Flp-In rtTA (Chtop_w/oExon5), siChtop, +Dox (Chtop mEx5 isof resc), Total frac, Bio rep 1; Mus musculus; RNA-Seq
For nuclear/cytoplasmic fractionation experiments N2a Flp-In rtTA cells were grown for 72 hours in a 6-well dish as described above. After 72 hours, N2a cells were harvested through addition of 200 µL Trypsin (0.05%) for ~10 minutes until most cells could be dislodged from the plate with gentle shaking. After trypsinization, cells were resuspended in 500 µL of DMEM growth media and the total volume transferred to a 1.5 mL microcentrifuge tube. To ensure similar number of cells in each sample for lysis, cells were then counted using an automated cell counter (Millipore, Scepter Handheld Automated Cell Counter) and equal number of cells transferred to a new microcentrifuge tube corresponding to the lowest cell count within each experiment. Next, cells were pelleted by centrifugation (300 rcf for 3 minutes) and then washed two times with 500 µL PBS and samples placed on ice for all remaining steps. 20% (100 µL) of the sample was removed as a “total” sample, centrifuged (300 rcf for 3 mins at 4 °C), and cell pellets resuspended in 200 µL of pre-chilled RIPA buffer containing freshly added 1mM DTT and 1mM PMSF and stored on ice. The remaining sample (400 µL) was pelleted (300 rcf for 3 mins at 4 °C), and 200 µL pre-chilled RLN buffer (recipe from QIAGEN: “Purification of cytoplasmic RNA from animal cells using the RNeasy Mini Kit”) containing 50 mM Tris·Cl (pH 8.0), 140 mM NaCl, 1.5 mM MgCl2 and 0.5% (v/v) Nonidet P-40 with freshly added 1mM DTT and 1mM PMSF was added slowly, and mixed by carefully inverting the tube and gentle pipetting. Samples were left for 5 minutes until homogenous and then centrifuged (300 rcf for 3 mins at 4 °C) to pellet the nuclei and cell debris. The lysate (~200 µL total) was removed and centrifuged at maximum speed (17,000 rcf for 3 mins at 4 °C) to remove any undigested cell debris and lysate transferred to a new tube (the “cytoplasmic” fraction). The remaining pellet (containing nuclei) were gently washed again with pre-chilled 200 µL RLN buffer as described above and pelleted again by centrifugation (300 rcf for 3 minutes at 4 °C). Pellets were then resuspended in 200 µL of RIPA buffer (the “nuclear” fraction). All samples were sonicated (2x15 pulses) to help sample homogenization before the lysates were divided with approximately one-third of the sample (~80 µL) taken for protein analysis and approximately two-thirds (~120 µL) taken for RNA analysis. For protein samples, Laemmli buffer was added and samples stored at -80 °C until western blots performed as described above. RNA was purified directly (same day) from the RLN buffer lysates using the RNeasy Mini Kit (QIAGEN) as recommended by the manufacturer and samples prepared for sequencing. RNA samples (250 ng) were enriched for mRNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490L, NEB) and subsequent libraries obtained using the NEBNext Ultra II Directional RNA library prep kit (E7760L, NEB) with some modifications. Specific modifications include; 1) fragmentation time was reduced from 15 minutes at 94°C to 7.5 minutes, 2) During the first strand cDNA synthesis reaction, the incubation times were increased from from 15 to 50 minutes at 42°C and 3) fragments were size selected using Ampure XP Beads (17.5ul for removing large fragments and 10ul to retain fragments without dimers).