GSM8268594: whole_brain, WT,very_old_mouse_3_1; Mus musculus; RNA-Seq
Mice were culled via cervical dislocation and brains were dissected, with the olfactory bulbs and cerebellum being removed.The desired area of the brain 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.