NextSeq 500 paired end sequencing; RNA-seq of human cerebromicrovascular endothelial cell line hCMEC/D3 or primary human brain vascular pericytes stimulated with TNF, or S. pneumoniae, in monoculture or in coculture, or stimulated with conditioned media from monocyte-derived macrophages (MDM) stimulated with S. pneumoniae, all compared to unstimulated controls
For the monoculture cell transcriptome experiments, the cells were cultivated in 6 well plates and stimulated for 4 hours on day 6. The supernatants were then removed, and the stimulated cells lysed with 600l of buffer RLT (Qiagen). This was stored at -80C until extraction.For the cocultured cells, the surface of basal chambers of 6 well transwell plates (Corning) were coated with 400ul of 15g/ml poly-l-lysine overnight, then washed with PBS.  HBVP were seeded onto the surface of the basal chambers at 5000/cm2 and allowed to adhere for 24 hours.  6.5mm diameter polycarbonate membrane transwells with 3m pores were coated with 100l of 75g/ml rat collagen-I solution for 2 hours, then washed with PBS.  The HBVP seeding solution was then replaced with 400ul of complete EBM-2 in each basal chamber and hCMEC/D3 cells were seeded at 50 000/cm2 onto the apical surface of the transwell membrane.   The coculture transwells were stimulated for 4 hours on day 6. The transwells were then moved into a new 6 well plates and supernatants were  removed from both transwells and basal chambers.  The cells were then lysed separately, each with 600l of buffer RLT (Qiagen). This was stored at -80C until extraction. RNA samples in buffer RLT were thawed and extracted using RNeasy Mini Spin Columns according to the manufacturers instructions. RNA was eluted in 30l of molecular grade water (Sigma) and quantified using a Nanodrop 2000 spectrometer.Contaminating DNA was removed through treatment with Turbo DNA-Free (Thermo), as per the manufacturers instructions RNA samples were provided to the PGU facility at UCL for library preparation and RNA sequencing using the Kappa Hyperprep kit (Roche) with polyA selection of mRNA and fragment size of 300-350 base pairs.   During this process a unique index adapter sequence is added to each sample to enable multiplexing and then identification and sorting of libraries during computational analysis.