title: Caco-2 3D culture with Sakei
design: For global transcriptomic assays, Caco-2 cultures (ca. 106 cells) were incubated for 30 minutes in the presence of L. monocytogenes or L. sakei 1 (MOI of 100), before total RNA extraction. At time 0, spent DMEM was removed from wells with confluent growth (2-D) and from tubes with beads (3-D). It was replaced by 2 ml aliquot of fresh DMEM (condition 1); 1 mL of fresh DMEM + 108 cells (1 ml) of L. sakei 1 (condition 2); 1 ml of fresh DMEM + 108 cells (1 mL) of L. monocytogenes (condition 3). The cultures were incubated for 30 min at 37oC with 5% CO2. By the end of incubation, culture media were removed, 1 ml of RNAprotect Cell Reagent (Quiagen, USA) was added and samples were stored at 4oC for later RNA extraction. Total RNA was extracted using RNeasy Plus Micro Kit (Qiagen) according to the manufacturers protocol. RNA integrity was preliminary assessed with NanoDrop, Qubit (fluorimetric assay) and agarose gel electrophoresis.  RNA samples were sent for sequencing at the Multi-users Laboratory of the Superior Agriculture School Luiz de Queiroz, located in the city of Piracicaba  So Paulo, Brazil. At the third-part laboratory, the RNA integrity was double checked using an Agilent 2100 bioanalyzer per manufacturers specifications. Sample concentrations were normalized, and 1,000 ng was used for library preparation. Libraries were prepared with the RNA extracts following the RNA TrueSeq preparation guide from Illumina, low sample protocol. This step converts the mRNA in total RNA into a library of template molecules suitable for subsequent cluster generation and DNA sequencing using the reagents provided in the Illumina TruSeq RNA Sample Preparation Kit v2. Briefly, the poly-A containing mRNA molecules are purified using oligodT attached magnetic beads, the purified mRNA is fragmented into small pieces, the cleaved RNA fragments are copied into first strand cDNA, second strand cDNA synthesis follows and adapters are ligated. In the final steps, products are purified and enriched with PCR to create the final cDNA library. Libraries prepared were diluted, pooled and sequenced in the sequencer HiSeq 2500 (Illumina, San Diego, CA, USA), using the HiSeq SBS v4, with paired readings of 100 bp (2x101). The 3-D culture conditions were done as independent triplicates and a single run of 2-D culture was performed only for comparison purpose.