title: GSM9034715: B9642-Hindbrain; Mus musculus; RNA-Seq
library_construction_protocol: Tissue samples were stored at -80°C. For tissue lysis and washing of nuclei, sample sections were added to 1 mL Nuclei PURE lysis buffer (Sigma, NUC-201) and thawed on ice. Samples were then Dounce homogenized with PestleAx20 and PestleBx20 before transfer to a new tube, with the addition of additional lysis buffer. Following incubation on ice for 15 minutes, samples were then filtered using a 30 mM MACS strainer (Fisher, NC0642012), centrifuged at 500xg for 5 minutes at 4°C using a swinging bucket rotor (Sorvall Legend RT, Thermo Fisher), and then pellets were washed with an additional 1 mL cold lysis buffer and incubated on ice for an additional 5 minutes. A 1.8M sucrose buffer was created and 1.8ml was mixed with 1ml of cell suspension. 1ml of sucrose buffer was deposited to ultra centrifuge tube (Fisher, NC9157569) then the cell suspension was layered on top (2.8ml). The suspension was then centrifuged at 30,000xg (17,800 rpm) for 45min at 4C, Rotor SW55ti. Supernatant was then aspirated off, and pellet was resuspended in 1ml of wash buffer (Nuclei Pure storage buffer, Sigma NUC-201) and spun at 500xg for 5 min at 4°C. Supernatant was aspirated and 0.5ml of wash buffer was added for resuspension Library preparation and NovaSeq Sequencing: Libraries were prepared according to 10xGenomics protocol for Chromium Single Cell 3' Gene Expression V3.1 kit. NovaSeq sequencing was performed according to illumine NovaSeq 6000 protocol.