GSM3290583: CT18; Mus musculus; RNA-Seq
Mice were sacrificed by acute cervical dislocation. Mice heads were chilled in the ice-cold dissection buffer for 10 seconds immediately. Brains were dissected out from the chilled head rapidly. The SCN region were microdissected in ice-cold dissection buffer by microsurgery forceps and knives according to the mice brain atlas . The SCN region tissues from 5 mice were pooled together. Tissues were digested by papain for 45 min and DNase I for 3min in digestion buffer with bubbling (95% O2, 5%CO2) under 37℃ incubation. Digestion was neutralized by adding 2ml digestion neutralizing buffer (KCl 5.33 mM, NaHCO3 26.19 mM, NaCl 117.24 mM, NaH2PO4 1.01mM, Dextrose 5.56mM, DL-AP5 50μM, DNQX 20μM, TTX 0.1μM, 5%(v/v) FBS, 5%(v/v) Trehalose). Tissue pieces were dissociated into single-cell suspension by gently pipetting using Pasteur pipettes with polished tips. Cell suspension was passed through 40μm filter and centrifuged (200g) for 7min at 4℃. The precipitate was re-suspended in 1ml Drop-seq buffer. Cell concentrations were determined with hemocytometer and diluted into 150 cell/μl with Drop-seq buffer. cDNA libraries were prepared using drop-seq protocols described by Macosko et al (2015).
GEO
GSM3290583