Fetal sheep microglia culture protocol was adapted from an established human adult that was modified to include a myelin removal step following the high-speed centrifugation.
{"action": "develop", "output": "", "reagent": ["", ""], "device": [""]}
flasks at a concentration of 2 × 10<sup>6</sup> cells
{"action": "microwave", "output": "", "reagent": ["", ""], "time": [""]}
1% penicillin/ streptomycin, , in which microglia grow best.56 Cells were allowed to incubate for seven days at 37°C, 5% CO<sub>2</sub>, followed by a media change by centrifugation .
{"action": "allow", "output": "", "time": ["seven days"], "temperature": ["37°C"]}
Cells were continued to incubate for seven more days with 5% DMEM at 37°C, 5% CO<sub>2</sub>, before the floating cells were collected. #####%%%%% output "E coli O127, B8" are used in line12
{"action": "continue", "output": "E coli O127, B8", "time": ["seven more days"], "temperature": ["37°C"]}
After carefully collecting the floating microglia to avoid contamination with astrocytes , the cells were incubated in 24-well plate at 1 x 10<sup>5</sup> cells/mL with 5% DMEM for another 4-5 days, .
{"action": "collect", "output": "floating microglia", "reagent": ["astrocytes"]}
then treated with , from E coli O127, B8 for 6h #####%%%%% output "media 0.5ml TriZol" are used in line14
{"action": "treat", "output": "media 0.5ml TriZol", "reagent": ["E coli O127, B8"], "time": ["6h"], "concentration": [""]}
Cell conditioned media 0.5ml TriZol per well added for RNA extractionwere collected for cytokine analysis, . #####%%%%% output "RNA extractionwere" are not used
{"action": "do", "output": "RNA extractionwere", "reagent": ["media 0.5ml TriZol"]}
The cell morphology was documented with light microscopy.
{"action": "examine", "output": "", "device": ["light microscopy"]}
Another portion of floating cells was plated onto Lab-Tek 8 well chamber glass slide \(Thermo Scientific) . #####%%%%% output "RNAseq" are used in line20
{"action": "plate", "output": "RNAseq", "container": ["floating cells"], "device": [""]}
**RNAseq approach** The overall experimental design was divided into three phases: sequencing, ). #####%%%%% output "TRIzol Reagent (Life Technologies)" are used in line22
{"action": "separate", "output": "TRIzol Reagent (Life Technologies)", "reagent": ["RNAseq"]}
RNA extraction : Total RNA was extracted from cultured microglia using TRIzol Reagent \(Life Technologies). #####%%%%% output "a RNA Nano Chip (Agilent RNA 6000 Nano Chips)" are used in line24
{"action": "extract", "output": "a RNA Nano Chip (Agilent RNA 6000 Nano Chips)", "reagent": ["cultured microglia", "TRIzol Reagent (Life Technologies)"]}
RNA quantity was established by using a RNA Nano Chip \(Agilent RNA 6000 Nano Chips) with Agilent 2100 BioAnalyzer. #####%%%%% output "Illumina TruSeq RNA Sample Preparation v2 kit (Illumina)" are used in line26
{"action": "establish", "output": "Illumina TruSeq RNA Sample Preparation v2 kit (Illumina)", "reagent": ["a RNA Nano Chip (Agilent RNA 6000 Nano Chips)"], "device": ["Agilent 2100 BioAnalyzer"]}
RNAseq libraries were prepared using Illumina TruSeq RNA Sample Preparation v2 kit \(Illumina)
{"action": "prepare", "output": "", "reagent": ["Illumina TruSeq RNA Sample Preparation v2 kit (Illumina)"]}
quality control was performed on the BioAnalyzer.
{"action": "perform", "output": "", "device": ["BioAnalyzer"]}
Single-end 50-bp sequencing was performed at high throughput on an Illumina HiSeq2500 at the CHU Ste-Justine Core Facility Sequencing Platform. #####%%%%% output "Single-end" are not used
{"action": "perform", "output": "Single-end", "device": ["an Illumina HiSeq2500"]}