GSM9454279: slender bsf, 0hrs after differentiation, replicate 2; Trypanosoma brucei brucei; RNA-Seq
Cells were harvested from biological triplicates, resuspended in 1 ml pre-warmed PBS containing 10 % FCS. The cells were washed twice with pre-warmed PBS to remove FCS in 1 ml of pre-warmed PBS 15 min prior to sorting, Calcein-AM violet was added at a final concentration of 1 µM to label viable cells. Live cells were sorted using a FACS Aria III cytometer (BD Biosciences). 1000 cells per replicate were deposited into single wells of a 48 well-plate containing 2.6 µl of 1x lysis buffer (Takara) and 0.01 µl of RNase inhibitor (40 U/µl; Takara). Sorting was performed in triplicates for each timepoint, and plates were immediately placed on ice and stored at -80°C. Bulk RNA-sequencing analysis was performed according to scripts and online resources published by Berry et al., 2021 using RStudio (version 4.02).