title: Bulk RNA-seq of HBMEC, iBMEC-like cells, and ETS-iBMEC in Response to Plasmodium falciparum Egress Products [for a manual submission]
design: Bulk RNA-seq of HBMEC, iBMEC-like cells, and ETS-iBMEC in Response to Plasmodium falciparum Egress Products [for a manual submission]
library_construction_protocol: This protocol describes the culturing and experimental conditions for hbmec and ibmec used for bluk RNASeq. iBMEC and hBMEC culture for bulk RNASeq   Human brain microvascular endothelial cells (hBMEC) hBMECs (Cell Systems, lot 376.11.04.01.2F) were cultured according to the manufacturer's instructions. Cells were seeded at 3 × 104 passage 7 hBMECs per well in 24-well plates and grown with daily media changes for 3 days.   iBMEC (DOX-induced and not DOX-induced)  Human IMR90-4 induced pluripotent stem cells (iPSCs) (WiCell, lot WB65316) were cultured following the manufacturer's instructions. This genetically modified cell line contains a PiggyBac insertion of a Tet-On motif, enabling DOX-inducible upregulation of the ETS transcription factors ETV2, ERG, and FLI1. The B6 clone was used for differentiation and experiments. Differentiation was performed according to Park et al., Nat Commun, 2019, with the following modification: On day 6, DOX induction was applied to generate the DOX cell line, while no induction was applied for the noDOX cell line. On day 8, the resulting cell populations were purified through subpassaging and washing on collagen-fibronectin-coated plates. Cells were then seeded into 24-well plates at 7.5 × 104 DOX-induced and 1 × 105 non-DOX-induced cells per well.   P. falciparum egress media exposure Parasite egress media was prepared as described in Long et al., bioRxiv, 2024, and applied to the respective samples at a concentration of 5 × 107 egressed parasites per mL for 8 or 24 hours.    Total RNA isolation At the end of the experimental timeline, each sample was fixed in Buffer RLT, and total RNA was extracted using the RNeasy QIAGEN Mini Kit, following the manufacturer's instructions, including on-column DNase digestion. RNA quality and concentration were assessed using the Tapestation system (Agilent Technologies). The final samples were adjusted to ~100 ng total RNA and stored at -80°C. This protocol prepares stranded mRNA-seq libraries from total RNA using the NEBNext RNA Ultra II Kit with polyA enrichment. NEBNext RNA Ultra II Kit for bulkRNA library prepIndividually barcoded stranded mRNA-seq libraries were prepared from high quality total RNA samples (~100 ng/sample) using the New England Biolabs NEBNext RNA Ultra II Kit combined with the polyA-enrichment module, according to the manufacturers instructions with 12 PCR cycles,implemented on the liquid handling robot Beckman i7. Obtained libraries that passed the QC step were pooled in equimolar amounts; 650 pM solution of this pool was loaded on the Illumina sequencer NextSeq 2000 and sequenced uni-directionally, generating 1980 million reads, each 80 bases long.