title: GSM3330375: Microglia 10 days post LPC 2; Mus musculus; RNA-Seq
library_construction_protocol: Mice were perfused with ice cold PBS with 10% sodium citrate. Lesions were isolated, manually dounced, and cells isolated by Percoll gradient. Microglia were FACS isolated, first excluding dead cells, then gating on Cd11b+ Ly6G- CD3- Cd45lo cells. 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).