GSM6067820: sfp1 KO; Schizosaccharomyces pombe; RNA-Seq
The cell pellets were resuspended in a preheated (65°C) solution composed of 25 µL of 10 % SDS and 300 µL of acid phenol: chloroform. Acid washed glass beads and bead buffer (75 mM NH4OAc and 10 mM EDTA, pH 8) were then added to the cell resuspension. The samples were vortexed for 1 minute, incubated at 65°C for 1 minute and this process was repeated for 3 times. After cell disruption, the samples were incubated at 65°C for 10 minutes and vortexed for 1 minute. The aqueous phase was recovered through centrifugation at 16000 ×g for 15 minutes and mixed with 250 µL of preheated (65°C) bead buffer. The samples were further subjected to centrifugation (16000 ×g, 4 °C, 15 minutes) and the supernatant was transferred to prechilled bead buffers with isopropanol (Wako). After centrifuged at 22000 ×g, 4°C for 30 minutes, the pellets were washed with RNase-free 70% ethanol. The RNA pellets were dried and dissolved in 30 µL of DEPC water. RNA libraries for RNA-seq were prepared using NEBNext ultra directional RNA library prep kit for Illumina following manufacturer's protocols.