GSM4605023: NPC-Con5; Mus musculus; RNA-Seq
All procedures were carried out at 4°C. Mice were perfused, cerebella were chopped and homogenized using a Dounce homogenizer in 2ml cold Medium A (HBSS+ 15mM HEPES+0.05% glucose+ 1:500 DnaseI), filtered through 100µm cell strainer, rinsed with 5ml Medium A and centrifuged at 340g for 5 mins. For myelin removal, the precipitate was resuspended in 25% standard isotonic percoll (25% Percoll in PBS, diluted with Medium A) and centrifuged at 950g for 20 mins. CD11b+Tmem119+ microglia (~20,000 per sample) were FACS sorted into 500µl RLT buffer (Qiagen) and stored on -80°C. RNA was extracted from isolated microglia using RNeasy Plus Micro kit (Qiagen) and the quality assessed by Agilent 2100 Bioanalyzer (Stanford PAN facility). About 1ng RNA was converted to cDNA and amplified for 12 cycles using SMART-seq v4 Ultra Low input RNA kit for sequencing (Takara Bio USA, Inc.) according to manufacturer's instructions. Amplified cDNA was then purified by immobilization on AMPure XP beads. Purified cDNA was normalized and tagmented for 5 mins using Nextera XT DNA library prep kit (Illumina). Unique indexes were then added to each sample, which were then amplified for 12 cycles. cDNA was purified using AMPure XP beads and quality assessed by Advanced Analytical Fragment Analyzer (Stanford PAN facility). Samples were then normalized and pooled together and sequenced on Illumina Novaseq 6000 (Novogene Corporation Inc.) to obtain 150bp paired-end reads.
GEO Accession
GSM4605023