GSM6323473: microglia,14month,female,LPS; Mus musculus; RNA-Seq
The brain without cerebellum were cut into pieces and then dissociated in 8U/mL papain lysis buffer containing 125 U/mL DNaseI) for 20 minutes at 37 ℃ with mild shaking. This process was then terminated by adding a 10% ovomucoid in L15 culture medium (2 mg/mL). After digestion, cell clusters were removed by filtering through 70 um nylon cell strainers (Faclon). Myelin and cell debris were removed from the mixed cell through the density gradient centrifugation in 37% Percol (SolarBio). after acquiring the mixed brain cells, the mixed cell were resuspended using FACS buffer. Next, mixed cells were stained with antibody against CD45 (1:100, clone 30-F11, BD Pharmingen, cat: 553080) and CD11b (1:100, clone M1/70, BD Pharmingen, cat: 557657). Death cells were labeled by 7-AAD (1:80, BD Pharmingen, cat: 559925). Followed by cell staining, CD45+CD11b+7-ADD-microglia were sorted and collected using BD FACSAria III (BD Biosiences). The harvested cells were then used for scRNA-seq, bulk RNA-seq or ATAC-seq based on different purpose. The single-cell library was constructed using the ChromiumTM Controller and ChromiumTM Single Cell 3' Reagent Version 2 Kit (10x Genomics, Pleasanton, CA).Briefly, single cells, reagents and Gel Beads containing barcoded oligonucleotides were encapsulated into nanoliter-sized GEMs (Gel Bead in emulsion) using the GemCode Technology. Lysis and barcoded reverse transcription of polyadenylated mRNA from single cells were performed inside each GEM. Post RT-GEMs were cleaned up and DNA were amplified. cDNA was fragmented and fragments end were repaired, as well A-tailing was added to the 3' end. The adaptors were ligated to fragments which were double sided SPRI selected. Another double sided SPRI selecting was carried out after sample index PCR. The final library was quality and quantitated in two methods: check the distribution of the fragments size using the Agilent 2100 bioanalyzer, and quantify the library using real-time quantitative PCR (QPCR) (TaqMan Probe). The final products were sequenced using the Illumina Hiseq 4000.