Transcription_Start_Site_data_from_mixed_asexual_blood_stages_of_P__falciparum_strain_3D7-sc-3620
Transcription_Start_Site_data_from_mixed_asexual_blood_stages_of_P__falciparum_strain_3D7
In eukaryotes, the chromatin architecture has a pivotal role in regulating all DNA-related processes. For P. falciparum, the causative agent of human malaria, the nucleosome landscape of the extremely AT-rich intergenic regulatory regions is largely unexplored. With the aid of a highly-controlled MNase-Seq procedure we reveal how positioning of nucleosomes provides a structural and regulatory framework to the transcriptional unit. We observe strong positioning of nucleosomes around splice sites that could aid co-transcriptional splicing events. In addition, nucleosome depleted regions are apparent hallmarks of transcription start sites (TSS) and might support preinitiation complex assembly. Moreover, we reveal nucleosome occupancy dynamics on strong TSSs during intraerythrocytic development, which anti-correlate with gene expression changes and we observe a characteristic nucleosome architecture of functional - but not inert - TGCATGCA DNA motifs. Collectively, these findings highlight the regulatory capacity of the nucleosome landscape of this deadly human pathogen.  This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
RNA-seq with tags to identify 5' ends of transcripts. Total RNA was isolated from in vitro cultured P. falciparum 3D7 parasites (using standard methods), and polyA+ RNA was isolated using magnetic oligo d(T) beads. Superscript II reverse transcriptase was used to synthesise first strand cDNA using oligo d(T) primers and in the presence of template switching oligos, which had the same sequence as those in the Smart-seq2 protocol (Picelli et al., 2013). The resulting cDNA was fragmented, made into RNA-seq libraries using an amplifcation-free protocol (as described in Spence et al., 2013) and sequenced using 150 bp PE read on an Illumina MiSeq. Reads were mapped using SMALT (which allows reads to be soft-clipped), and reads mapping as proper pairs were selected. Reads containing the sequence corresponding to the template-switching oligo were identified using BLAT, and were used to infer the 5' ends of transcripts.