GSM7876849: bsf_2; Trypanosoma brucei; RNA-Seq
After natural infection, skin equivalents were cultured for another 4 h, 12 h, 24 h, and 7 d, respectively. In addition, freshly collected MCFs obtained from tsetse flies and cultured BSFs were used. Skin equivalents were dissociated and single viable parasites were sorted based on their tdTomato and Calcein AM signal with a FACSAria III cytometer (BD Biosciences) into 48-well plates containing 2.6 µl of 1x lysis buffer (Takara) supplemented with 0.01 µl of RNase inhibitor (40 U/µl; Takara). Immediately after sorting, cells were placed on ice for 5 min and stored at -80 °C. After natural infection, skin equivalents were cultured for another 4 h, 12 h, 24 h, and 7 d, respectively. In addition, freshly collected MCFs obtained from tsetse flies and cultured BSFs were used. Skin equivalents were dissociated and single viable parasites were sorted based on their tdTomato and Calcein AM signal with a FACSAria III cytometer (BD Biosciences) into 48-well plates containing 2.6 µl of 1x lysis buffer (Takara) supplemented with 0.01 µl of RNase inhibitor (40 U/µl; Takara). Immediately after sorting, cells were placed on ice for 5 min and stored at -80 °C. Library preparation and sequencing was carried out as described previously (Muller et al., 2018). Briefly, 0.2 µl of a 1:20 x 106 dilution of ERCC Spike-in Control Mix 1 (Thermo Fisher Scientific) were added to the lysates of single parasites and libraries were prepared using SMART-Seq v.4 Ultra Low Input RNA Kit (Takara) using a quarter of the reagent volumes recommended by the manufacturer. 27 cycles were used for PCR amplification and cDNA was purified using Agencourt AMPure XP beads (Beckman Coulter) using 15 μl of elution buffer (Takara). Library quantification was performed with a Qubit 3 Fluorometer with dsDNA Hs Assay kit (Life Technologies) and the quality of the libraries was assessed using a 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent). 0.5 ng of cDNA was subjected to a tagmentation-based protocol (Nextera XT, Illumina) using a quarter of the recommended volumes, 10 min for tagmentation at 55 °C and 1 min extension time during PCR amplification. Libraries were pooled and sequencing was performed in paired-end mode for 2 × 75 cycles using Illumina's NextSeq 500.