GSM7593973: Hindbrain pain neuronal ensemble SNI with brush; Mus musculus; RNA-Seq
Mouse hindbrains (~ -7.3- -5.2 mm posterior to the bregma) were rapidly dissected under sterile conditions and stored in -80°C. Hindbrains from the same treatment were pooled together for nuclear isolation. In breif, pooled brain tissues were homogenized (2ml Dounce homogenizer) in 1 ml freshly prepared lysis buffer, filtered (40 µm cell strainer), and centrifuged at 300g for 5 min (4°C). The pellet was gently resuspended in 500 µl 1xPBS (1% BSA and 0.2 U/µl SUPERaseIn RNase Inhibitor) and loaded on top of 900 µl 1.8M Sucrose Cushion Solution (Sigma, NUC-201). After 45 min of ultracentrifuge (13,000 g, 4°C), the nuclei were resuspended in 500 µl FACS buffer with the addition of 2.5 µl of 5 mM nuclear marker DRAQ5 (ThermoFisher). Library was constructed folowing the manufacturer's protocol (10x Genomics Chromium Single Cell 3' Reagent Kit v3)