Raw transcriptomic data of Trypanosoma cruzi supplemented with hemin and hemoglobin

Introduction This dataset has the data to determinate the impact of free heme (added as hemin) and Hb supplementation on the Trypanosoma cruzi epimastigote transcriptome. Using Next Generation Sequencing and alignment to the T. cruzi Dm28c 2018 genome, we analyzed transcriptomic changes 4 and 24 hours after heme re-supplementation, compared to heme-starved parasites. Transcriptome data can be analyzed by comparing all samples to time 0 (heme-starved), or heme re-supplementation sample against non-supplemented samples at each time point (4 and 24h). The dataset also includes raw data of Western blot and qPCR, to design and validate the heme starvation conditions used for the transcriptomic assay, and of alphaFold predicted structures of the new identified protein TcCRAL/TRIO. Value of the data Trypanosoma cruzi, the causative agent of Chagas disease, cannot synthesize heme and relies on scavenging it from its hosts and vectors. In the triatomine insect midgut, epimastigotes are exposed to hemoglobin (Hb) and heme (free heme) product of Hb degradation. This dataset represents the first transcriptomic analysis of Trypanosoma cruzi epimastigotes exposed to two distinct heme sources—hemin and hemoglobin—at early time points (4 and 24 hours) following heme restitution after heme starvation. Our work provides novel insight into the immediate gene expression changes triggered by physiologically relevant heme sources, capturing early regulatory events and potentially transient responses that are critical for parasite adaptation. Heme restitution induced a rapid, but mild, global shift in the epimastigote transcriptome. This dataset offers a valuable resource for understanding heme sensing and metabolism in T. cruzi, and may help identify novel molecular targets for therapeutic development. Content of the dataset and file organization The dataset includes 73 files organized into four main sections (folders), corresponding to the different types of experiments performed: SECTION A- RNAseq Raw data The files correspond to the different determinations. For example “BR1-t24-0 heme_1” refers to a sample from biological replicate (BR) 1, taken at time 24 h (t24), from a culture with 0 uM heme (0 heme), part 1. Due to the size of the data, the data of each determination is divided into 2 files (part 1 and part 2). SECTION B- RT-qPCR Raw Data The file contains the Cq values ​​for each of the determinations for each of the validated genes. SECTION C- Raw data of TcCRAL-TRIO structure prediction by Alphafold The files contained here correspond to the files used to initiate the structure prediction job by AlphaFold, files of each of the five (0 to 4) structures of TcCRAL/TRIO predicted by Alphafold, and the files of the superposition. SECTION D- Western Blot The files correspond to the images of the Western Blot tests performed, one photo per treatment tested.. Instrument -or software- specific information needed to interpret the data RNAseq: The quality of raw reads obtained from the RNA-seq assay was assessed using FastQC (Andrews, 2010). Adapter trimming and low-quality base removal were performed using sickle. Reads from each library were aligned to the Trypanosoma cruzi Dm28c 2018 reference genome (available at TriTrypDB: https://tritrypdb.org) using Bowtie2 (Langmead & Salzberg, 2012; doi:10.1038/nmeth.1923), with the "end-to-end" alignment mode. The resulting SAM alignment files were converted to BAM format and indexed using SAMtools (Li et al., 2009; doi:10.1093/bioinformatics/btp352) for downstream processing and to obtain mapping statistics. Gene-level quantification was performed using featureCounts (Liao et al., 2014; doi:10.1093/bioinformatics/btt656), restricting counting to coding sequences (CDS). A global count matrix was generated, and differential gene expression analysis (DEG) was conducted in R using the DESeq2 package (Love, et al., 2014, DOI: 10.1186/s13059-014-0550-8). Normalized counts were visualized using heatmaps generated with the pheatmap package. Additional data exploration and visualization were carried out using R packages including dplyr, ggplot2, RColorBrewer, ggrepel, stringr, hrbrthemes, GGally, and viridis. RT-qPCR Raw Data: Instrument used: CFX Opus 96 Real-Time PCR System (#12011319 - Bio-Rad Laboratories, Hercules, CA, USA) and Eppendorf Mastercycler epGradient (#5341, Eppendorf, Hamburg, Germany). The files were exported to .xls. TcCRAL/TRIO structure prediction by Alphafold: Structural visualization of TcCRAL/TRIO predicted structure, model overlay, and comparison with the crystal structure of S. cerevisiae Sfh5 (PDB ID: 6W32; Khan, et al., 2021, DOI: 10.7554/eLife.57081) were performed using ChimeraX (Meng, et al., 2023, DOI: https://doi.org/10.1002/pro.4792). Variable List: -Time -Culture condition (heme, hemin, hemoglobin) The specific information for each variable is described in the Methodological information section. Specialized formats or other abbreviations used: Hb: hemoglobin. qRT-PCR: quantitative real-time PCR. RB, R or BR: Biological replicate R1, R2, R3: Biological Replica 1, Biological Replica 2, Biological Replica 3. ScSFH5 Saccharomyces cerevisiae Sec-Fourteen Homolog 5 t0: time 0. 0 hours. 0 h. t4: time 4. 4 hours. 4 h. t8: time 8. 8 hours. 8 h. t10: time 10. 10 hours. 10 h. t24: time 24. 24 hours. 24 h. TcHRG: Trypanosoma cruzi Heme Responsive Gene WB: Western blot NH, hem, Hb: No heme (NH) addition, or addition of 5 µM hemin (hem) or 1.25 µM hemoglobin (Hb) after 48 hours of heme starvation.