title: RNA-seq of triple cultures of neurons, astrocytes and microglia treated with blood clot conditioned media, lipopolysaccharide, and Nrf2-activating drug CDDO-TFEA.
design: RNA-seq of triple cultures of neurons, astrocytes and microglia treated with blood clot conditioned media, lipopolysaccharide, and Nrf2-activating drug CDDO-TFEA.
library_construction_protocol: We thawed frozen primary human astrocytes (-190oC; Caltag Medsystems, SC-1800) and plated them in in poly-D-lysine-coated T175 flasks. They were incubated with human astrocyte medium (Caltag Medsystems, SC-1801) for a maximum of 7 passages using Trypsin EDTA (Sigma, T3924). For experiments, these were seeded in 24-well tissue culture plates (Greiner, 662160) at 3-5x105 cells per well and left for 3 days until 80-90% confluency was achieved.  We dissected the cortices of Sprague Dawley rat embryos of embryonic day 20.5 on ice cold DM+K media and removed the hippocampi. We performed enzymatic digestion at 37oC using papain for 40 minutes, with papain solution changed midway. We then washed the cortices twice with 37oC DM+K and then twice more with 37oC 1% neurobasal-A (neurobasal-A-medium [NBA; Gibco, 10888-022]; 1% v/v rat serum [Harlan SeraLab, R-0100B], 1x antibiotic-antimycotic, 2% v/v B-27 supplement [Invitrogen, 17504-044], 1mM L-glutamine) before triturating in 1% NBA. We diluted this cell suspension in 37oC OptiMEM I+ (OptiMEM I [Gibco, 31985-047], 0.36% w/v D-[+]-glucose, 1x antibiotic-antimycotic) to 14mL per cortical hemisphere used and plated 0.5ml on top of each well containing human primary astrocytes, having first aspirated the human astrocyte medium. After 2.5h, we aspirated the OptiMEM I+ and debris and replaced with 1mL fresh 37oC 1% NBA with 4.8μM Cytosine β-D-arabinofuranoside hydrochloride (AraC; Sigma, C6645). These co-cultures of human astrocytes and rat neurons were incubated until DIV2 whereupon we further 1% NBA with AraC to each well. At DIV 4, we exchanged 1mL media for 0% NBA (NBA, 1x antibiotic-antimycotic, 2% v/v B-27 supplement, 1mM L-glutamine) with 0.18% w/v D-[+]-glucose.  Mouse pups (P0-P4) were decapitated and brain tissue placed in ice cold dissection media (DM+K; sodium sulfate, potassium sulfate, magnesium chloride, calcium chloride, HEPES, phenol red, D-(+)-glucose, kynurenic acid). We used 20 mins papain digestion (papain 36,000 USP units/mL [Merck Millipore,5125]) at 37oC before washing and triturating in DMEM solution (Dulbecco's modified Eagle's medium [Gibco, 31966-021], 10% foetal bovine serum [Invitrogen, 10108-165], 1x antibiotic-antimycotic [streptomycin, penicillin, amphotericin B; Thermo Fisher, 15240062]). We plated this cell suspension onto poly-D-lysine (BD Bioscience, 354210) coated T175 flasks and placed these in a humidified incubator (37oC, 5% CO2, 20% O2) for 2.5h before replacing the media with fresh DMEM solution and allowing growth to confluency for 12-14 days. After 12-14 days of growth, we placed mixed glial cultures on an orbital shaker at 150rpm for one hour at 37oC to detach microglia. We collected the media and centrifuged at 150xG for five minutes. We resuspended pellets in 0% NBA to achieve a concentration of 2x105 live cells per mL. 1mL of this was plated on top of DIV7 neuron-astrocyte cultures, having first removing the media from each well. These were incubated for two days to produce co-cultures of WT or KO primary mouse microglia, WT human astrocytes and WT rat neurons for experimentation.  We isolated RNA immediately on termination of each experiment using High Pure RNA Tissue kits (Roche 12033674001) according to manufacturer's instructions.  We prepared TruSeq stranded mRNA-seq libraries (Illumina).