GSM8309179: RISP_KO_treatment_Naïve_ageGroup_Adult_05; Mus musculus; RNA-Seq
For FACS of microglia for RNAseq and microglial enumeration, single cell suspension from whole brain were initially incubated in 1:50 Anti-mouse CD16/32 (biolegend 101302, clone 93) and, subsequently, stained with the following antibodies: 1:100 Anti-Mouse CD45.2-PacBlue (ebioscience 48-0454-82, clone 104) and 1:200 Anti-mouse CD11b(Mac-1)-APC (ebioscience, 50-0112-80, clone M1/70) in 200µL final volume of FACS Buffer. Viability was assessed with addition of DAPI 3 minutes before collection on flow cytometer (Fisher, D1306 0.1µg/mL final concentration). Immediately after sorting microglia were centrifuged at 500xg for 10 minutes at 4°C then resuspended in 350µL RLT plus buffer (Qiagen) and stored at -80°C until nucleic acid purification with the AllPrep DNA/RNA micro kit (Qiagen) for downstream analysis. RNA quality and quantity were assessed using TapeStation 4200 High Sensitivity RNA tapes (Agilent), and RNA-seq libraries were prepared from 5-10ng of total RNA using the SMARTer Stranded Total RNA-seq Kit v2 (Takara Bio). For analysis of allele status, libraries were prepared using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (NEB E7370L). Library QC was then performed using TapeStation 4200 High Sensitivity DNA tapes (Agilent). Dual-indexed libraries were pooled and sequenced on a NextSeq 500 instrument (Illumina) for untreated RISP-/- and wild-type, age-matched animals, or a NextSeq2000 instrument for all other samples (Illumina) 75 or 100 cycles, respectively, single-end, to an average sequencing depth of 27.5 million reads per sample.