GSM9195890: GR CUT&RUN on P14 NR V1 GR-Con astrocytes, replicate 2; Mus musculus; OTHER
In vivo: 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. After dissecting 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). 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. 175,000 DRAQ5+/GFP+ or DRAQ5+/GFP- events were collected into 1 mL of cold CUT&RUN Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.2% Tween-20, 0.5 mM spermidine, 0.1% bovine serum albumin [BSA], 1X PIC). In vitro:  3T3 cells were washed twice with cold PBS and then scraped in NE1 lysis buffer (20 mM HEPES pH 7.9, 10 mM KCl, 0.1% Triton X-100, 3 mM MgCl2, 0.5 mM spermidine, 1X PIC, 10 mM sodium butyrate). Nuclei were rotated for 10 min at 4°C and centrifuged at 500g for 10 min at 4°C. The nuclei pellet was then resuspended in 1 mL of CUT&RUN Wash Buffer. Takara SMARTer ThruPlex DNA-seq Kit