RNA extraction. RNA from patient tissue samples stored in RNAlater was extracted using the Qiagen RNeasy Plus Mini kit (Qiagen). Briefly, tissue samples were homogenized in the Qiagen RLT Lysis Buffer (Qiagen) and processed using the Polytron Homogenizer (Kinematica). The homogenized lysate was passed through genomic DNA eliminator columns (Qiagen), subsequently applied to RNeasy spin columns, followed by several washes according to the manufacturer’s instructions and eluted in nuclease-free water.

NanoString analysis. The nCounter custom code set was designed for P. falciparum that included differentially expressed genes to distinguish specific stages including asexual parasites (circulating rings and sequestrating trophozoites/schizonts) and sexual stages (gametocyte rings and immature and mature gametocytes) as defined from our previous transcriptional network analysis study (33). In total, there are 161 genes representing asexual circulating stages, 147 genes representing asexual sequestering stages, 26 genes representing gametocyte rings, 27 immature gametocytes, and 29 mature gametocyte genes. The remaining set was not annotated for any of these stages. A total of 456 genes were included in this custom probe set including housekeeping genes. For NanoString analysis of tissue samples, 500 ng of purified total RNA was used as input. Briefly, RNA from each sample was allowed to hybridize with reporter and capture probe set at 65°C for 20 hours according to the nCounter gene expression assay protocol (NanoString Technologies). After several washes in the preparation, RNA-probe complexes were immobilized to nCounter cartridge followed by scanning in the nCounter Digital Analyzer. Data were normalized on the basis of background subtraction, and expression of housekeeping genes using nSolver Analysis software (NanoString Technologies) according to guidelines and statistical analysis was performed. The normalized data were included in table S1.

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