title: GSM4726615: spinal cord microglia G93A (end stage)_rep1; Mus musculus; RNA-Seq
library_construction_protocol: Mice were all females and were deeply anesthetized using Xylazine (Rompun 0.2%) and Ketamine (Imalgene 500) and transcardially perfused with ice-cold 0.1M PBS.     For sciatic nerve peripheral macrophages (MP), two whole left and right sciatic nerves (from the gastrocnemius muscles to the spine vertebrae) were collected and pooled, except for the macrophages at the pre-symptomatic stage where four sciatic nerves coming from two mice of the same age were needed to recover enough cells, and for spinal cord microglial cells (MG), whole spinal cords were flushed using 0.1M PBS. Tissues were chopped in DMEM-F10 (1:1, Life Technologies) culture medium supplemented with 10% Fetal Calf Serum (FCS, low endiotoxins, Life technologies). Tissues were passed through a 100μm nylon filter (Falcon). Myelin was removed using a percoll gradient (30-37-70% percoll, GE healthacare) in 0.1M PBS. The whole procedure was performed at 4°C and did not use enzymatic digestion/ incubation at 37°C to avoid ex-vivo activation of the cells. Briefly, 1.6 ml of 70% Percoll solution were laid at the bottom of 5ml polypropylene tubes (Falcon), gently covered by 1.6 ml of 37% Percoll. Cells were homogenized in 30% Percoll solution and carefully laid on top and centrifuged (without break) for 40 minutes at 800g at 4°C. The 30% percoll layer containing myelin was removed and cells were recovered at the interface between the 37% and the 70% Percoll layers. Cells were resuspended in 0.1M PBS, 0.5% BSA (Sigma Aldrich), 2mM EDTA (Life Technologies).    Microglial cells (MG) or peripheral macrophages (MP) were isolated using an anti-CD11b magnetic microbead-coupled antibody and MS columns (Miltenyi Biotec) according to manufacturer's instructions. Between 1.0x10^5 and 1.8x10^5 microglial cells were recovered from spinal cords (one spinal cord from one animal per sample, see Publication for more details) and between 3x10^4 and 5x10^4 macrophages were recovered from sciatic nerves (two sciatic nerves from one animal per sample except for the pre-symptomatic stage where 4 sciatic nerves from two mice were pooled, see Publication for more details) depending on the genotype and disease stage of the mice. For RNA isolation, cells were centrifuged at 300g at 4°C and kept at -80C until further use.    RNA was extracted from spinal cord microglial cells and from sciatic nerve macrophages using the Nucleospin RNA XS kit (Macherey Nagel) with a final RNA elution in 10μl. Microglial cell RNA concentration was determined with a RNA 6000 pico kit (Agilent Technologies) on an Agilent Bioanalyzer. Although the number of microglial cells and macrophages and therefore the total quantity of RNA obtained from the two cell populations, were different, to compare microglial cells and macrophages, we used the same protocol and the same kits and we therefore used only a fraction of the microglial RNA obtained. One ng of microglial cell RNA (from total RNA of 1.50-7ng/microglial sample) and whole total eluted RNA of sciatic nerve macrophages (RNA quantities for macrophage were too low to be measured, but estimated from the number of cells, in comparison to microglia) were used to perform cDNA synthesis using the Smart-Seq v4 Ultra Low Input RNA kit for sequencing (Clontech laboratories) with linear pre-amplification. Library preparation was made with 150 pg of amplified cDNA for every sample (microglia and macrophages) using the Nextera XT DNA Library Preparation Kit (Illumina). Eight samples were run with a 400 million cartridge to reach an average coverage of 50 million reads per sample (75bp, paired-ends). Sequencing was performed using the NextSeq 500 high Output Kit V2 on a Illumina NextSeq 500. Library preparations and sequencing were performed by the ICM iGenSeq core facility.