GSM7090271: M2,replicate 2, scRNAseq; Mus musculus; RNA-Seq
0.1-0.3 g of brain tissue in each group was washed with 1xPBS (Thermo Fisher), minced and homogenized in a glass homogenizer containing 3 mL of Buffer A (250mM sucrose (Sangong), 10mg/mL BSA (Thermo Fisher), 5mM MgCI2 (Sinopharm), 0.12U/μL RNasin (Promega), 0.06U/mL SUPERasenTM RNase Inhibitor (ThermoFIsher), 1x Protease Inhibitor (CST)), and beat 10 times on ice with a large gap and a small gap pestle in turn. The mixture was filtered through a 100 um cell sieve filter, and the filter was washed twice with 1 mL and 750 uL of Buffer A. After the addition of Triton X-100 (Thermo Fisher), a non-ionic surfactant with a concentration of 0.5%, the mixture was further homogenized in a pestle and whipped 50 times. After the mixture passed through a 35um cell sieve membrane, following steps were performed in sequence: centrifuged at 500 g at 4°C for 5 min in turn, the supernatant was discarded, the pellet was resuspended in 1 mL of citric acid solution (0.25 M sucrose (Sangong), 25 mM citric acid (Sigma), 1ug/mL of Hoechst 33342 (Thermo Fisher)), centrifuged at 500 g at 4°C for 5 min, discarded the supernatant, centrifuged at 3000 g at 4°C for 5 min, resuspend the pellet in 1 mL Buffer B, and centrifuge, resuspended in 200uL Buffer B (320mM sucrose (Sangon), 10 mg/mL BSA (Thermo Fisher), 3mM CaCl2 (Sinopharm), 2mM MgAc2 (Sinopharm), 10mM Tris-HCl (Sigma), 0.1mM EDTA (Sigma), 1mM DTT (Invitrogen), 1 mM DTT (Invitrogen), 0.12U/μL RNasin (Promega), 0.06U/mL SUPERasenTM RNase Inhibitor (ThermoFIsher), 1xProtease Inhibitor (CST)), and centrifuged at 20.000g for 3 min at 4°C. Finally, the pellet was stained with trypan blue (Thermo Fisher) and counted by CountStar. Single nucleus were run on a 10× Chromium system (10 × Genomics) and then through a library preparation by LC Sciences, following the recommended protocol for the Chromium Single Cell 30 Reagent Kit (v2 Chemistry). Libraries were run on the HiSeq4000 for Illumina sequencing.