GSM9195999: RNA-seq on P28 NR V1 astrocyte GR-Con nuclei, replicate 2; Mus musculus; RNA-Seq
Mice were deeply anesthetized with isoflurane. Sections of 1 mm spanning V1 were collected in an adult mouse brain matrix (Alto) on ice, and V1 was microdissected. For bulk RNA-seq, tissue samples were immediately flash-frozen in a dry ice ethanol bath and stored at -80°C prior to nuclei extraction. After thawing samples, V1 tissue was transferred to a 1.5 mL tube containing 350 μL of cold homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM tricine-KOH pH 7.8 supplemented with 1X PIC, 1 mM DTT, 0.15 mM spermine and 0.5 mM spermidine). For RNA experiments, 0.3 U/μL (bulk RNA-seq) of Protector RNase inhibitor (Sigma) was added. The tissue was dounced in a 2 mL glass homogenizer (Sigma D8938) with a loose pestle for 20 strokes. Samples were supplemented with 0.3% IGEPAL CA-630 and further dounced for an additional 10 strokes with a tight pestle. Homogenate was filtered through a small 40-μm strainer (pluriSelect 43-10040-50) into a 1.5 mL tube before diluting 1:1 with homogenization buffer and adding DRAQ5 (Abcam ab108410) at a 1:500 dilution. Nuclei were then sorted by GFP and/or DRAQ5 signal using the Sony SH800S Cell Sorter (purity mode) with a 100-μm sorting chip. For bulk RNA-seq, 80,000 DRAQ5+/GFP+ events were collected into 350 μL of cold RLT buffer (Qiagen 74004) supplemented 1:100 with β-mercaptoethanol. After sorting nuclei into RLT buffer, RNA was extracted using the RNeasy Micro Kit (Qiagen 74004). RNA-seq libraries were prepared from 10 ng RNA per sample using the SMARTer Stranded Total RNA-Seq Kit v2 (Takara 634413), according to the manufacturer's instructions. Libraries were sequenced paired-end on an Illumina NextSeq 500 using a 75-cycle kit.