title: PC_2098_induced_48h_2
organism: Trypanosoma brucei
description: Protocols: Procyclic cells (EATRO 1125 strain) with a Tet-repressor construct (pHD1313) and an inducible RBP10-myc construct (pHD2098) was used. The cells were grown at 27oC in MEM-Pros medium supplemented with 10% heat inactivated fetal bovine serum. To convert procyclic (PC) to bloodstream (BS) forms, RBP10-myc was induced for 48 hours using tetracycline (200ng/ml), the cells were pelleted, resuspended (2x105 cells/ml) in HMI-9 medium and then incubated at 37oC with 5% CO2. For RNA-seq, RNA was isolated from two biological replicates after RBP10 expression for 24h and 48h; uninduced procyclic cells served as control. Two additional RNA samples from re-differentiated bloodstream forms (newly converted BS) and starting EATRO 1125 bloodstream (BS AnTat1.1_2098) cells were included. Trifast reagent (Peqlab, GMBH) was used for total RNA isolation. 5µg of total RNA was depleted of ribosomal RNA (rRNAs) using RNAse H (5U) in presence of 50-base DNA oligos complementary to trypanosome rRNAs. After DNAse I treatment, RNA was purifed using RNA Clean & Concentrator kit (Zymo Reseach, USA). RNA-seq was done at the CellNetworks Deep Sequencing Core Facility at the University of Heidelberg. For library preparation, NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs Inc.) was used according to the manufacturer's instructions.