| Name |
Description |
Order Applied |
| GSM7982876 channel 1 extraction |
Following the completion of MEA experiments, neurons from 6 replicate wells were lysed in the well using TRIzol (Invitrogen) and pooled RNA was isolated using manufacturer protocols. Purity and concentration of RNA were confirmed using Nanodrop, and Zymo RNA Clean & Concentrator-25 was used on any samples that did not pass initial quality control (260/280 > 2, 260/230 > 1.8, minimum concentration 50 ng/µl). Libraries prepared via Illumina-compatible kit by Azenta/Genewiz using PolyA selection |
1 |
| GSM7982876 channel 1 treatment |
Human i3 cortical neurons were generated as previously described (Wang, Ward et al. 2017, Fernandopulle, Prestil et al. 2018). In brief, human iPSCs reprogrammed from a wild type male containing a doxycycline-inducible NGN2 integrated into the AAVS1 safe harbor locus were cultured on matrigel-coated (Corning) plates in supplemented Essential 8 medium (Gibco). Once iPSC cultures reached confluence, they were split and induced toward neuronal differentiation with doxycycline-containing induction media (DMEM/F12-Gibco, N2, & NEAA-Invitrogen) for 3 days. On DIV 3, induced neurons were lifted and plated PEI/Laminin-coated Axion CytoView MEA plates. Neurons were plated at a density of 2x105 cells per ml for MEA experiments. Cells were matured in cortical maturation media (BrainPhys-Stem Cell Technologies, B-27-Gibco, BDNF, NT3, and laminin) for an additional 18 days prior to experiment initiation. |
1 |
| GSM7982876 channel 1 growth |
Elvitegravir and raltegravir were resuspended in dimethysulfoxide (DMSO, Veh). Working stocks were prepared via serial dilution in DMSO to ensure all doses (0.1 µM, 1 µM, and 10 µM) contained the same amount of DMSO. i3 neurons were treated with INSTIs beginning on DIV 18 and maintained for 9 days for MEA experiments. Fifty percent media changes every other day were supplemented with an additional half dose of drug to ensure any removed was replaced, but no drug metabolism was assumed. For LPS + ATP samples, uninfected iMg cultures were treated with 1 ng/ml E. coli LPS for 23.5 hours beginning on DPI 8, with a 30 minute addition of 2.5mM ATP prior to supernatant collection. i3 neurons were treated with supernatants at a ratio of 1:4 in cortical maturation media beginning on neuronal DIV 19 and maintained for 4 days. |
1 |