title: GSM7213985: miR-132 OE, Run1, scRNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: Mice received an overdose of pentobarbital and were perfused with ice-cold artificial CSF (aCSF). The aCSF was composed of (in mM): 93 NMDG, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 10 MgSO4, 0.5 CaCl2, and was adjusted to pH 7.4 and equilibrated in 95% O2 5% CO2. After perfusion, right hippocampi were microdissected and placed in cold Hibernate A Low Fluorescence (BrainBits), supplemented with 5 μM Actinomycin D (Cat#A1410-5MG, Sigma-Aldrich). Hippocampi were dissociated enzymatically using the Adult Brain Dissociation Kit, mouse and rat (Cat#130-107-677, Miltenyi Biotec) for 15 min at 37°C, with 5 μM Actinomycin D added. Subsequently mechanical dissociation was performed, cells were passed through a 70 µm cell strainer and washed with 10 ml ice-cold D-PBS supplemented with 0.5% BSA. Next, cells were centrifuged for 10 min at 300 g at 4°C and myelin and debris removal was performed according to manufacturer's instructions. Afterwards, cells were resuspended in D-PBS (0.5% BSA) and labeled with the eBioscience™ Fixable Viability Dye eFluor™ 780 (1:2000, Cat#65-0865-14, Thermo Fisher Scientific, irreversibly labels dead cells) and in run 2, 4, 5 also with Anti-mouse ACSA-2-APC (1:20, Cat#130-116-142, Miltenyi Biotec) and BV421 Rat Anti-CD11b (1:10, Cat#562605, BD). Cells were incubated for 30 min in the dark at 4°C, washed with 1 ml D-PBS (0.5% BSA) and spun down for 10 min at 300 g at 4°C. Subsequently, cells were suspended in 200 μl D-PBS (0.5% BSA) for FACsorting. Only alive cells were sorted, as dead cells were excluded based on their staining with the eFluor™ 780 dye. Neurons were sorted based on YFP expression. For astrocyte isolation, ACSA-2 positive microglia were excluded by selection based on both CD11b and ACSA-2 expression. According to the run, an Influx (BD) or MACSQuant Tyto (Miltenyi Biotec) were used for sorting and different cell types were isolated. 10000-20000 cells were sorted per sample and further processing was conducted following manufacturer's instructions (CG000183 ChromiumSingleCell3 v3 for RUN 1, 2, 3 and CG000204 ChromiumNextGEMSingleCell3 v3.1 for RUN 4, 5). Briefly, cells were loaded onto the Chromium Single Cell Chip B (RUN 1, 2, 3) or Chromium Next GEM Chip G (RUN 4, 5) for GEM formation and after consecutive steps of reverse transcription, cDNA clean-up and amplification, DNA libraries were generated. Libraries were loaded in an equimolar pool for sequencing on the HiSeq4000 or NovaSeq6000 platform (Illumina) aiming for 50000 reads/cell.