RNA sequencing of blood brain barrier spheroids exposed to either Neisseria meningitidis or Borrelia bavariensis in in vitro conditions

Neisseria meningitidis and Borrelia bavariensis are the causative agents of invasive meningococcal disease and Lyme neuroborrelosis. Before infecting the brain parenchyma the bacterial pathogens should cross the human blood-brain barrier (BBB). To understand the response of BBB against the invading pathogens, in vitro BBB spheroid model was generated by co cultivating human brain microvascular endothelial cells, pericytes and astrocytes in low adhesion conditions. Spheroids (N= 36) were infected with either N. meningitidis (6 x 10e4, MOI 1:4) or B bavariensis(1.5 x 10e5, MOI 1:10) for 3h. Two separate control groups of spheroids (N=36) without any infection were included in the study. After 3h of treatment (infection/no infection), 12 spheroids were pooled to form one biological sample and such 3 biological replicates per treatment were sampled for total RNA extraction. Thereafter, 12 cDNA libraries were prepared using QuantSeq FWD 3'mRNA Library Prep Kit (Lexogen). Second strand of cDNA library was synthesized using the UMI Second Strand Synthesis Mix (USS, Lexogen) containing 6 nucleotides long Unique Molecular Identifiers (UMIs). The double stranded cDNA libraries were amplified in 17-19 PCR cycles using i5 Unique Dual Indexing Add-on Kit (Lexogen). Illumina NextSeq 500 was employed to sequence the libraries having a read lengths of single-end 75?bp producing about 10 million reads per library. To carryout gene expression analysis, Bioconductor R-package - DESeq2 v1.20.0 (Love et al., 2014) was used where in, featureCounts tool v1.6.3 (Liao et al., 2013) was used to enumerate the genes. A minimum threshold of adjusted p-value < 0.05 and log2fold-change (log2FC) = ± 1 was set to considered the gene as differentially expressed (DEG).