GSM8169681: IgG_2_low; Mus musculus; RNA-Seq
Brains were dissociated into single cells with the Neural Tissue Dissociation Kit (Miltenyibiotec, 130-092-628). Briefly, the brains were chopped using a razor, resuspended with HBSS, and peletted by centrifugation. The pellet was resuspended and incubated with the enzyme P mix at 37°C along with the transcriptional and translational inhibitors Actinomycin D (Cell Signaling Technology, cat. #15021, 5uM final concentration) and Anisomycin (Cell Signaling Technology, cat. #2222, 2uM final concentration) respectively. Samples were homogenized by trituration using a 5 ml serological pipette and incubated with Enzyme A at 37°C. Afterwards, the brains were triturated with a 2ml serological pipette, washed with DPBS, passed through a 100uM filter and centrifuged at 300xg for 10 min. To remove the excess myelin, cells were resuspended in 0.9M sucrose and centrifuged at 850g for 35 min. Cells were washed with the FACS buffer (1x PBS, 1% BSA, 2mM EDTA) and stained with SYTOX™ blue (ThermoFisher) to distinguish live cells. At this point, cells from 1 male and 1 female from each treatment group were pooled and processed together. (Data from individual animals were later demultiplexed based on sex-specific gene expression, see below.)  Flow cytometry revealed that ~1% of the live brain cells from the control IgG group were AF647 positive, compared to ~30% and ~20% for the ATV-TfR and ATV-CD98hc groups, respectively. We divided the AF647 positive cells from the ATV-TfR treatment group into tertiles according to fluorescence intensity, e.g. we binned them into low, medium, and high categories and applied the same FACS gates to samples from all treatment groups. In addition, AF647-negative cells were collected, generating 4 sub-populations (negative, low, medium, high intensity) for each pool. Single cells from 24 samples (3 genotypes x 2 replicates x 4 FACS bins) were captured: ~60,000 cells were sorted into a 1.5 mL centrifuge tube. To concentrate the single-cell suspensions, each sample was pelleted by centrifugation at 200xg for 10 minutes in a swing bucket rotor at 4°C, and supernatant was removed leaving only 43.3 µL volume for resuspension. The entire volume of cells was then added to the RT master mix and loaded onto Chromium Next GEM Chip G microfluidic channels per manufacturer's protocol (10X Genomics, CG000315 Rev C). One sample (IgG, negative fluorescence) was lost due to a fluidic wetting issue, leaving 23 samples for library preparation and sequencing analysis. Post GEM–RT Cleanup was performed using a Dynabeads cleanup mix via magnetic purification, and cDNA was generated using 12 cycles of amplification followed by cDNA purification using SPRIselect reagent. A quarter volume of the cDNA generated was used as input for 3' gene expression library construction starting with fragmentation, end repair and A-tailing, followed by Illumina adaptor ligation and sample index PCR, with SPRIselect bead purification performed in between each step. Library quantity and quality were assessed with High Sensitivity D1000 ScreenTapes (Agilent 5067-5584). The libraries were pooled for quality control in equimolar ratios and analyzed by shallow sequencing on an Illumina MiSeq instrument (Reagent Kit v3, Illumina, MS-102-3001) to estimate the total number of cells captured for each sample. Based on this estimate, individual libraries were pooled targeting 20,000 reads per captured cell and  the sequenced deeply on an Illumina NovaSeq 6000 instrument, S2 cartridge, Paired end (28X10X10X90) by SeqMatic (Fremont, CA, USA).