GSM6637185: cortex, KO, mouse3; Mus musculus; RNA-Seq
Brains were collected from 6–7-week-old female mice at the same time of day for each animal. Mice were culled via cervical dislocation and brains were dissected, with the olfactory bulbs and cerebellum being removed. Hippocampi from both hemispheres and the remainder of the left hemisphere (without the hippocampus) were collected in ice-cold HBSS (w/o Ca2+ and Mg2+; 14175-053; Gibco) with 5% trehalose (T0167; Sigma Aldrich) and 30 µM actinomycin D (A1410; Sigma Aldrich) and were finely minced using a 22A scalpel. Brains were digested using the Adult Brain Dissociation Kit (130-107-677; Miltenyi Biotec), with the following modifications: (i) tissues were dissociated as described in the “manual dissociation” section of the Neural Tissue Dissociation Kit protocol (130-092-628; Miltenyi Biotec), (ii) enzymatic digestions were performed at 35°C, (iii) half the concentration of enzyme P was used, (iv) actinomycin D was used to limit dissociation-induced transcriptional changes,  (v) 5% trehalose was added in all buffers to increase cellular viability, (vi) cell clusters were removed by filtration through prewetted 70 μm (352350; Falcon) and 40 μm (352340; Falcon) cell strainers, (vii) erythrocyte and myelin debris removal steps were omitted during dissociation steps, and (viii) all centrifugations were performed at 200 g at 4°C. After dissociation, cells were collected in PBS with 0.2% BSA before being sorted on a Sony SH800 cell sorter. Gates were chosen based on forward and side scatter to exclude myelin debris, erythrocytes, and doublets. Non-viable cells were excluded based on Draq7 and/or DAPI staining (Draq7high and/or DAPIhigh cells were classified as non-viable). single cells were processed through the Chromium Single Cell Platform using the Chromium Next GEM Single Cell 3' GEM Library and Gel Bead Kit (v3.1 chemistry, PN-1000121, 10x genomics) and the Chromium Next GEM Chip G kit (PN-1000120) and processed following manufacturer's instructions.