title: GSM8001580: APOE2 astrocytes; Mus musculus; RNA-Seq
library_construction_protocol: 6 months old mice were anaesthetized using CO2 chamber and perfused with ice-cold 1x DPBS (Gibco, #14190-144). Each mouse brain, excluding cerebellum and olfactory bulbs were dissected and placed in FACS buffer (1x DPBS, 2% FCS and 2 mM EDTA) + 5 μM Actinomycin D (ActD, Sigma, #A1410-5MG) for transcriptomics. Brains were mechanically and enzymatically dissociated using Miltenyi neural tissue dissociation kit P (Miltenyi, #130-092-628) supplemented with 5 μM ActD. Next, samples were passed through a 70 μm strainer (BD2 Falcon), washed in 10 ml of ice-cold FACS buffer + 5 μM ActD and spun at 300 rcf for 15 minutes at 4 °C. Note that 5 μM ActD was kept during collection and enzymatic dissociation of the tissue to preserve transcriptional state and prevent artificial induction stress-response genes during tissue dissociation (Perocchi et al., 2007; Marsh et al., 2022). ActD was removed from the myelin removal step to prevent toxicity derived from long-term exposure. Following dissociation, myelin was removed by resuspending pelleted cells into 30% isotonic Percoll (GE Healthcare, #17-5445-02) and centrifuging at 300 rcf for 15 minutes at 4 °C. Accumulating layers of myelin and cellular debris were discarded and Fc receptors were blocked in FcR blocking solution (1:10, Miltenyi, #130-092-575) in cold FACS buffer for 10 minutes at 4°C. Next, cells were washed in 5 ml of FACS buffer and pelleted cells split into two suspensions (one each for astrocyte and microglia sorting), and incubated with the following antibodies: for microglia- PE-CD11b (Miltenyi, #130-113-806, 1:50), BV421-CD45 (BD, #563890, 1:500); for astrocytes- APC-ACSA2 (Miltenyi, #130-116-142, 1:200), BV421-CD11b (BD, #562605, 1:500), PE-O4 (Miltenyi, #130-117-357, 1:50); in cold FACS buffer for 30 minutes at 4 °C. TotalSeq™-A cell hashing antibodies (1:500, Biolegend) and viability dye (eFluor 780, Thermo Fisher Scientific, #65-0865-14) were also added to both fractions during the incubation step. After incubation, cells were washed, and the pellet was resuspended in 500 μl of FACS buffer and passed through 35 μm strainer prior sorting. For isolating microglia, cell suspension was loaded into the input chamber of MACSQuant Tyto Cartridge and cells were sorted on MACSQuant Tyto Cell Sorter at 4 °C, based on CD11b-positive and CD45-low populations. For isolating AAV-transduced astrocytes, cells were sorted on Aria Fusion, based on negative gating for O4 and CD11b populations, followed by positive gating for double-positive ACSA2 and mCherry populations. 20,000-30,000 cells of each cell type (astrocyte and microglia) from each mouse were diluted to a final concentration of 1000 cells/µl. All the samples were individually hashed using TotalSeq™-A cell hashing antibodies, 3000 cells/animal were pooled and loaded onto the Chromium Next GEM Chip G (PN #2000177). The DNA library preparations were generated following manufacturers' instructions (CG000204 Chromium Next GEM Single Cell 3' Reagent Kits v3.1). In parallel the hashtag oligo libraries were prepared according to manufacturers' instructions (BioLegend - TotalSeq™-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v3 3.1 Protocol) using 16cycles for the index PCR. From the 4 experimental groups (APOEKO, APOE2, AOE3, and APOE4), a total of 8 libraries (APOEKO_astrocytes, APOE2_astrocytes, APOE3_astrocytes, APOE4_astrocytes, APOEKO_microglia, APOE2_microglia, APOE3_microglia, APOE4_microglia), containing 22 biological replicates (n(APOEKO)= 2, n(APOE2)= 3, n(APOE3)=3, n(APOE4)= 3), were sequenced (service provided by BGI Genomics, China) targeting 90% mRNA and 10% hashtag oligo library (50,000 reads/cell) on a DNBSEQ-G400 (MGI Tech) platform with the recommended read lengths by 10X Genomics workflow.