GSM8336697: 1222_WT_Pos; Mus musculus; RNA-Seq
Mouse brain samples were rinsed in D-PBS, sliced into sagittal sections, placed into gentleMacs C-tubes, and processed for generation of single-cell suspensions using the Adult brain dissociation kit and gentleMacsTM Octodissociator system (#130-107-677 and #130-095-937, respectively, Miltenyi Biotech, San Diego, CA) (Chucair-Elliott et al., 2020; Ocanas et al., 2022). The single-cell suspensions were then immunostained for flow cytometric analysis of EGFP+ cell populations in the brain. The gating strategy of single cells was set to EGFP+/CD11b-/CD31+ for endothelial cells and EGFP+/CD11b+/CD31- for putative microglia. The antibodies used were anti-mouse CD11b: Vioblue (#130-113-810, clone REA592) and anti-CD31: APC (#130-111-355, clone REA784) (Milteny Biotec). Samples were analyzed with a MACSQuant Analyzer 10 Flow Cytometer (Miltenyi Biotec USA, Gaithersburg, MA) using MACSQuantify v2.13.0 software (Ocanas et al., 2022). A 488 nm (blue) laser with 525/50 and 585/40 filter combinations was used to gate on EGFP+cells within single cells (singlets) without auto-fluorescence interference, as previously performed for NuTRAP models (Chucair-Elliott et al., 2020; Ocanas et al., 2022). A 640 nm laser (red) with 655/730 nm filter and a 405 nm (violet) laser with 450/50 filter were used for detection of CD31 and CD11b, respectively. For cell sorting of endothelial cells, immunolabeled brain cell suspensions were loaded into the input chamber of a MACSQuant Tyto cartridge, to run through a MACSQuant Tyto Cell Sorter system (#130-103-931, Miltenyi Biotec) (Ocanas et al., 2022). A blue (488 nm) laser with B1 (525/50 nm) and B2 (585/40 nm) filter combinations was used to gate on eGFP+ cells without auto-fluorescence interference. Subsequent gating was based on CD31: APC fluorescence used a red (638 nm) laser and R1 (655-730 nm) filter and exclusion of CD11b: Vioblue fluorescence, using a violet (405 nm) laser and V1 (450/50) nm filter. After completion of the sort, the cells from the positive fraction chamber were collected in 0.5% BSA in D-PBS. The positive fraction chamber was washed twice using 450 µL of 0.5% BSA in D-PBS per wash and combined with the initial positive fraction collection. An aliquot of (10%) of the positive fraction was kept for analysis on the MACSQuant Analyzer 10 Flow Cytometer while the rest of the sorted sample was pelleted, along with an initial aliquot of input cell suspension (pre-sort) was resuspended in RNeasy Plus lysis buffer and subjected to RNA extraction using the AllPrep DNA/RNA Mini Kit (#80204, Qiagen), according to manufacturer's instructions. RNA was quantified with a Nanodrop One spectrophotometer (Thermo Fisher Scientific) and its quality assessed by HSRNA ScreenTape with a 4150 TapeStation analyzer (Agilent Technologies, Santa Clara, CA). The NEBNext Ultra II Directional Library Prep Kit for Illumina (#NEBE7760L; New England Biolabs Inc., Ipswich, MA) was used with 25 ng of total RNA for the preparation of strand-specific sequencing libraries from TRAP-isolated RNA samples (input and positive fraction) according to manufacturer's instructions (Chucair-Elliott et al., 2020). Briefly, polyA containing mRNA was purified using oligo-dT attached magnetic beads. mRNA was chemically fragmented and cDNA synthesized. For strand-specificity, the incorporation of dUTP instead of dTTP in the second strand cDNA synthesis does not allow amplification past this dUTP with the polymerase. Following cDNA synthesis, each product underwent end repair process, the addition of a single 'A' base, and finally ligation of adapters. The cDNA products were further purified and enriched using PCR to make the final library for sequencing. Library sizing was performed with HS RNA ScreenTape (#5067-5579; Agilent Technologies). The libraries for each sample from each mouse model were pooled at 2-4 nM concentration and sequenced in independent runs using an Illumina NovaSeq 6000 system (PE150bp) at the OMRF Clinical Genomics Facility.