GSM8996319: mtRNAP_0d; Trypanosoma brucei; RNA-Seq
A total of 1x108 cells were harvested by centrifugation at 2500 rpm for 8 min at room temperature. The pellet was washed with 1 ml PBS, transferred to a 1.5 ml tube and centrifuged again at 2500 rpm for 8 min at room temperature. After removing the supernatant, the cell pellet was resuspended in 1 ml TRIzol (Tri-Reagent, Merck, #9424). Subsequently, 200 µl of chloroform was added and the samples were vortexed vigorously. The mixture was then centrifuged at 12000 rpm for 15 min at 4°C. The upper aqueous phase (400 µl) was carefully transferred to a new tube containing 500 µl of ice-cold isopropanol, ensuring that the interphase was not disturbed. The tube was inverted several times to mix the solution. RNA was precipitated by centrifugation at 12000 rmp for 15 min at 4°C. Subsequently, the supernatant was removed and discarded. The RNA pellet was washed twice with 1 ml 70% ethanol followed by centrifugation at 7500 rpm for 5 min at 4°C. The ethanol was removed, and the tube was inverted and left open at room temperature to air-dry for at least 5 min. Finally, the RNA pellet was resuspended in 50 µl RNase-free water and incubated at 55°C for 5 min on a heating block. RNA concentration was determined using a Nanodrop spectrophotometer and RNA integrity was assessed by running 1 µg of RNA on a 1% agarose gel for 40 min at 100 V. RNA was stored at -80°C or used directly for DNase treatment and cDNA synthesis. Total RNA are processd into short fragments (150-200 bp) and treatet with kinase. Libraries are prepared using the QIAseq-miRNA kit