GSM5396875: T0; Mus musculus; RNA-Seq
Lateral ventricles CP of five adult mice were rapidly microdissected and pooled together in ice cold dissecting medium (HBSS supplemented with 0.6% glucose). Dissected CPs were settle down by gravity to aspirate the dissection medium and replace with fresh dissection media supplemented with 3 mM CaCl2 and 100 µg/ml collagenase D (Sigma-Roche, # 11088858001) and incubate at 37°C in 20% O2 and 5% CO2 on orbital shaker for 15 min. After the incubation, collagenase containing medium was aspirated and CPs were further incubate in papain using papain dissosciation system kit (Worthington, cat#LK003150) following the manufactured instructions. Dissociated CPs were finally centrifuged at 280 g for 5 min and resuspended in PBS. Next, 10 µl of single cells suspension was mixed with 10 µl of 0.4% trypan blue and automatically counted using Countess 3 automated cell counter (Invitrogen, cat#AMQAX2000). Approximately 7000 cells from each sample were loaded into Single Cell Chip B (10X Genomics,#PN-1000092) using the Single Cell 3' v3 reagent kit (10X Genomics, # PN-1000074) for gel bead emulsion generation in the Chromium system. Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3 PE sequencing, standard 10XGenomics RNA-seq