Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

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

ABSTRACT: 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).

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Homo sapiens

SUBMITTER: Amod Kulkarni

PROVIDER: E-MTAB-13401 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:Spheroids, near spherical multicellular aggregates, are one of the most common types of threedimensional (3D) cell cultures. Despite decades of implementation of spheroid technology in various fields of life science and medical research, no minimal information (MI) guidelines are available to cope with heterogeneity and to stimulate transparency. To cope with this unmet need, we assembled an international consortium to develop the MISpheroID knowledgebase (https://www.mispheroid.org) and interrogation revealed heterogeneity and lack of transparency in published spheroid-related experiments. This steered us to empirically evaluate the impact of cell line, culture medium type, spheroid formation method and spheroid size on complementary spheroid metrics (RNA fingerprints, presence of cell death, ATP content, glucose to lactate conversion, secreted protein signatures, circularity, size and cancer therapy response). We measured media-induced transcriptional variation in lung cancer (A549), colorectal cancer (HCT116), ovarium cancer (SKOV3) and glioblastoma (U87MG) spheroids using RNA sequencing (RNA-seq). These spheroids were formed in ultra-low attachment plates and cultured in 6 different media types (DMEM high glucose, DMEM/F12, RPMI1640, DMEM low glucose, EMEM and MEM) for 5 (HCT116) or 7 (A549, SKOV3 and U87MG) days. RNA extraction was performed on 2 spheroids per condition using the miRNeasy micro kit (217084, Qiagen, Hilden, Germany). RNA-sequencing libraries were prepared from purified RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Vienna, Austria) according to the manufacturer's instructions, using 27.5ng of RNA that was DNase treated using HL-dsDNA (Arcticzymes, TromsØ, Norway). The individual libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche, Pleasanton, CA, US) and equimolarly pooled. The pool concentration was measured with Qubit and 1.4pM with 1% PhiX was sequenced on a NextSeq 500 (Illumina, San Diego, CA, US) using a high-output 1x75 run. Reads were mapped to the human genome using Tophat and gene expression counts were generated using HTSeq. This data was used to perform Principal Component Analysis (PCA) and Gene Set Enrichment Analysis (GSEA).

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets