GSE173907
Next generation sequencing provides valuable insights into the global view of the transcriptome dynamics in Leishmania cells with depleted levels of profilin as compared to control cells.
Transcriptome analysis via high-throughput sequencing (RNA-Seq) captures significant changes in gene expression. The purpose of this study is to elucidate the transcriptomic changes in profilin deficient Leishmania promastigotes (LdPfn+/-) as compared to wild type control (LdPfn+/+). This research will enable us to gain insight into the role of  profilin in Leishmania cells. The data has been validated by qRT-PCR. Data analysis results are further supported by flow cytometry, immunofluorescence experiments and by western blot demonstrating a depletion in the protein expression of a eukaryotic translation initiation factor in the cells with depleted levels of profilin protein. Methods: Total RNA from three independent biological replicates from each of wild-type (LdPfn+/+) and profilin knockout (LdPfn+/-) Leishmania promastigotes was extracted and after library preparation, RNA sequencing was carried out on Illumina HiSeq 2500.  The sequence reads that passed quality filters were aligned to the Leishmania donovani (BPK282A1) genomic data obtained from TriTrypDB version 51 using  Bowtie2 (-x option). All analyses were carried out using the Tophat pipeline with the following versions: Tophat v2.1.1, Bowtie2 v2.3.5.1. The HTSeq version 0.12.4 (htseq-count -f option) was used to count the number of reads aligned to protein-coding genes. DeSeq tool was used for differential gene expression analysis between samples in protein-coding genes. qRT-PCR validation was performed using SYBR Green assay. Results: RNA-Seq datasets generated in this study with each sample control (LdPfn+/+) and profilin knockout (LdPfn+/-) have at least 30 million read pairs. RNA-seq data were aligned to the L.donovani genome (Leishmania donovani BPK282A1, NCBI taxon ID: 981087) and 8135 transcripts were identified in our data set. Data analysis revealed that a total of 254 genes were differentially expressed (DE) having a p-value <0.05. The data obtained from RNA-Seq have been validated by real-time PCR of 18 genes, 8 UP regulated and 10 down-regulated. Results of qRT-PCR analysis showed a strong correlation with the RNA-seq data. Inferences drawn from transcriptomic analysis were further validated by flow cytometry, immunofluorescence experiments and western blotting, revealing a critical role during the early phases of Leishmania cell division. Conclusions: Present study represents a comprehensive report elucidating the transcriptomic changes in profilin deficient Leishmania promastigotes (LdPfn+/-) as compared to wild type (LdPfn+/+). This RNA-Seq data along with validation by qPCR, flow cytometry experiments, and immunofluorescent microscopy experiments indicate that profilin might play a critical role during the early phases of Leishmania cell division. Overall design: mRNA profiles of wild type (LdPfn+/+) and profilin deficient (LdPfn+/-) Leishmania donovani promastigotes.