NextSeq 550 sequencing; The SPOC proteins DIDO3 and PHF3 co-regulate gene expression and neuronal differentiation - 3' RNA-seq
The SPOC proteins DIDO3 and PHF3 co-regulate gene expression and neuronal differentiation - 3' RNA-seq
Subcellular fractionation, RNA extraction and 3'end sequencing library preparation. For 3'end sequencing cells were fractionated into cytoplasm and nuclei and RNA was extracted separately. 10 million HEK 293 cells were mixed with 1 million MEF cells as a spike-in control, washed once in 1xPBS and resuspended in 1 mL hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 1 mM PMSF, 0.1% Triton, 1x protease inhibitors). Cells were incubated on ice for 5 min, then dounced in a prechilled 7 mL Douncer for 10 strokes with a tight pestle to release nuclei. After centrifugation at 228g, 4°C for 5 min the supernatant was mixed with 200 µL 5x RIPA buffer (250 mM Tris pH 7.5, 750 mM NaCl, 5% NP-40, 2.5% deoxycholate, 1 mM PMSF, 0.1% Triton, 1x protease inhibitors) and centrifuged for 10 min at 2800g, 4°C. The supernatant was kept as the cytoplasmic fraction. The pellet from the first centrifugation step was resuspended in 600 µL sucrose buffer 1 (0.25 M sucrose, 10 mM MgCl2, 1 mM PMSF, 1x protease inhibitors) and layered over 600 µL sucrose buffer 2 (0.35 M sucrose, 0.5 mM MgCl2, 1mM PMSF, 1x protease inhibitors). After centrifugation at 1430g for 5 min, 4°C, the pellet (nuclei) was resuspended in 1 mL TRI reagent and processed for RNA extraction as described above. For RNA extraction from the cytoplasmic fraction, 300 µL cytoplasmic extract were mixed with 900 µL TRI reagent LS for liquid samples (Sigma), 240 µL chloroform was added and the lysate was vortexed and centrifuged at max. speed (21130g) for 15 min at 4°C. The aqueous layer was transferred to a new tube and precipitated with 0.6 mL isopropanol. The RNA pellet was subsequently treated as described above. Library preparation for 3' end sequencing of polyadenylated mRNAs was performed using 3' mRNA-Seq Library Prep Kit FWD (Lexogen) according to the manufacturer's instructions.