title: NextSeq 500 sequencing; RNA sequencing of blood brain barrier spheroids exposed to either Neisseria meningitidis or Borrelia bavariensis in in vitro conditions
design: RNA sequencing of blood brain barrier spheroids exposed to either Neisseria meningitidis or Borrelia bavariensis in in vitro conditions
library_name: 3h_NK_rep3_s
library_construction_protocol: 12 blood brain barrier-spheroids were produced by seeding human brain microvascular endothelial cells, human pericytes and human astrocytes (1:1:1 ratio) in ELISA plates wells (4500 cells/well) precoated with 1% agarose (50 microliter) and 150 micro liters of EGM2 bullet kit (Lonza). Within 48h of incubation in humidified 5% Co2 incubator, spheroids were self assembled. All the spheroids were transferred to ultra low adhesion plates containing endothelial medium devoid of antibiotics. Next, each spheroid (one spheroid/well) was infected with either Neisseria meningitidis (Nm) or Borrelia bavariensis (Br) for 3h (in vitro infection in humidified CO2 incubator). Two separate negative control (NK) groups for each bacterial infection group were also maintained that received no infection. After 3h of infection, all the 12 spheroids were pooled as one sample in microcentrifuge tube, one quick wash with PBS was performed and the sample was stored in -80 freezer until total RNA extraction.                Total RNA was extracted using RNeasy Mini Kit (Qiagen), which includes on-column DNase digestion as described by the manufacturer. Concentration of RNA was measured on NanoDrop and the integrity was assessed on 1% agarose gel electrophoresis.  RNA (500 ng) was reverse transcribed using oligodT primer included in QuantSeq FWD 3'mRNA Library Prep Kit (Lexogen, Austria). Half the recommended volume of OligodT primer (2.5 µL) was used to minimize the off-target products. RNA removal solution (RS buffer, Lexogen) was used on cDNA to eliminate any remaining RNA. Second strand was synthesized using the UMI Second Strand Synthesis Mix (USS, Lexogen) containing 6 nucleotides long Unique Molecular Identifiers (UMIs). UMI allows detection and removal of PCR duplicates. The double stranded cDNA libraries were amplified in 17-19 PCR cycles using i5 Unique Dual Indexing Add-on Kit (Lexogen). The quality and quantity of libraries was determined on Fragment Analyzer using DNF-474 HS NGS Fragment Kit (Agilent Technologies) and QuantiFluor dsDNA System (Promega), respectively.