GSM9196025: snRNA-seq on P21 V1 from DR astrocyte GR-KO animal, replicate 2; Mus musculus; RNA-Seq
Sections of 1 mm spanning V1 were collected in an adult mouse brain matrix (Alto) on ice, and V1 was microdissected. Tissue samples were immediately flash-frozen in a dry ice ethanol bath and stored at -80°C prior to nuclei extraction. 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). 1 U/μL (snRNA-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 DRAQ5 signal using the Sony SH800S Cell Sorter (purity mode) with a 100-μm sorting chip into a 1.5 mL tube (precoated with 30% BSA) containing 300 μL of cold ATAC-RSB supplemented with 1 mM DTT, 1 U/μL Protector RNase inhibitor, and 1% BSA (Sigma A8577-10mL). After collection, nuclei were centrifuged two times at 200g for 2 min in a swinging-bucket centrifuge at 4°C. The supernatant was then carefully aspirated from the surface of the liquid until approximately 40 μL remained. snRNA-seq libraries were prepared with the 10X Genomics Single Cell 3' Gene Expression Kit v4 (PN-1000686), following the manufacturer's instructions.