Sequencing of microglia during focal remyelination of the corpus callosum

The goal of the study was to determine the gene expression of microglia prior to the onset of remyelination (3 days post lysolecithin injection) and pro-regenerative microglia at the onset of remyelination (10 days post remyelination). Overall design: 3 mice were used per time point (3 dpl, 10 dpl). 10 week-old male C57Bl/6J mice were anaesthetised with isoflurane before being stereotaxically injected with 2 µl of 1% lysolecithin (LPC; vol/vol) into the corpus callosum. Mice were perfused with ice cold phosphate buffered saline with 10% sodium citrate. Lesioned corpus callosum was homogenised using a 2 ml Dounce and filtrated (250 µm filter; Pierce). Following a spin at 600 g for 5 min (with brake), cells were resuspended in 100 % fetal bovine serum (FBS) and 33 % Percoll (1:10), overlaid with 1 ml of 10 % FBS, and spun for 15 min at 800 g at 4 °C without brake. The cell pellet was washed in FACS buffer and spun for 10 min at 600 g at 4 °C, and incubated in anti-mouse CD16/32 Fc-block (Clone 93, BioLegend, 1:200) on ice for 10 min. Fluorescently-conjugated antibodies CD11b-PeCy7 (Clone M1/70, Invitrogen, 1:100), CD45-BV605 (Clone 30D11, Biolegend, 1:200), Ly6G-PerCP Cy5 (Clone 1A8, BioLegend, 1:200), and CD3-APC (Clone 17A2, BioLegend, 1:200) were applied on ice for 30 min. Following centrifugation and filtration (30 µm), cells were sorted by flow cytometry into FBS-coated Eppendorf tubes on ice. Cells were spun at 800 g for 5 min, resuspended in RLT Plus buffer with ß-mercaptoethanol, and centrifuged at 10,000 rpm for 2 min in QIAshredder tubes (Qiagen). RNA was extracted using the AllPrep DNA/RNA/miRNA kit (Qiagen) as per the manufacturer's instructions, and quantity/quality analysed using the Bioanalyser 2100 (Agilent) and RNA 6000 Pico kit (Agilent) as per the manufacturer's instructions. cDNA production/ library preparation was performed using the NuGEN Ovation RNAseq System v2 kit (NuGEN) by BGI (Hong Kong). End Repair Mix was added to the amplified cDNA and incubated at 20°C for 30 min. AxyPrep Mag PCR clean up kit (Axygen) was used to purify the end-repaired DNA, which was then combined with A-tailing Mix (Enzymatic) and incubated at 37°C for 30 min. Adaptors (Invitrogen) were ligated to the Adenylate 3' ends DNA, and incubated at 16°C for 16 h. Insert size was used to select the adaptor-ligated DNA fragments. Several rounds of PCR amplification with PCR Primer cocktail (Invitrogen) and PCR Master Mix (New England Biolabs) were performed to enrich the adaptor-ligated DNA fragments to produce the final library, purified using AxyPrep Mag PCR clean up kit (Axygen). The final library average molecule length was determined using the Bioanalyser 2100 using the DNA 1000 kit (Agilent) and was quantified by real-time qPCR (TaqMan probe). cBot (Illumina) was used to amplify the libraries to generate the cluster on the Flow Cell (HiSeq 4000, Illumina). An average of 40 million clean 100 paired-end reads (read lengths approximately 100 bp) were achieved per sample. Data was processed to remove adaptors and low quality reads from raw reads.