After 24-hour static or dynamic condition, hECs were removed from the LOC devices by very fast and gentle trypsinization and RNA was isolated using RNeasy Plus Micro Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Integrated gDNA eliminator spin column allows DNA depletion from RNA samples. RNA integrity (supplemental Figure 2) was checked using automated capillary electrophoresis (RNA Pico Sensitivity Assay, LabChip GX II Touch HT, Perkin Elmer, Waltham, MA, USA). RNA samples were stored at -80 °C until further analysis. Genome-wide gene expression profiling was performed using massive analysis of cDNA ends (MACE-seq) with RNA extracted from hECs co-cultured with PCs in static and dynamic conditions (3 and 5 biological replicates, respectively). A total of 8 libraries were constructed using the Rapid MACE-Seq kit (GenXPro GmbH, Frankfurt, Germany), according to the manufacturer's protocol. MACE-seq performs gene expression profiling by sequencing part of the 3' end of mRNA transcipts. While synthetizing one cDNA molecule from each mRNA transcript, MACE-seq can accurately quantify transcribed polyadenylated transcripts. Briefly, samples with 100 ng of purified RNA were used for library preparation. RNA was fragmented using GenXPro Fragmentation Buffer. Synthesis of cDNA was performed by reverse transcription using barcoded oligo(dT) primers containing TrueQuant unique molecular identifiers, followed by template switching. Library amplification was done using polymerase chain reaction (PCR), purified by solid phase reversible immobilization beads (Agencourt AMPure XP, Beckman Coulter, Brea, CA, USA) and subsequent sequencing was performed using a NextSeq platform (Illumina Inc., San Diego, CA, USA).
GEO Accession
GSM4709955