title: Ast_old_E
organism: Mus musculus
description: Protocols: Subventricular zones (SVZs) from 3-4-month-old or 19-22-month-old GFAP-GFP mice72 were dissected, processed, and sorted following the protocol described in Codega et al.73, with the addition of staining for cell types of other lineages (CD31 for the endothelial lineage and CD45.1 for the hematopoietic lineage). Briefly, SVZs were microdissected immersed in PIPES buffer pH 7.4, finely chopped, and digested for 10 min at 37°C in PIPES buffer pH 7.4 with 14 U/ml lyophilized papain (Worthington, LS003119) ordered within 1 week of use, layered and spun over a 22% Percoll gradient (Fisher, 45001747), and stained with 1:50 CD31-PE (eBioscience, 12-0311-81), 1:50 CD45.1-Brilliant Violet 605 (Biolegend, 110737), 1:400 CD24-eFluor 450 (eBioscience, 48-0242-80), 1:300 EGF-Alexa 647 (Molecular probes, E-35351), and 1:400 Prominin-1-biotin (eBioscience, 13-1331-80). Cells were then washed and stained with 1:1000 Streptavidin-PEcy7 (eBioscience, 25-4317). Propidium iodide was added for live/dead discrimination, and the cells were sorted on the BD FACS Aria with a 100 μm nozzle at 13 psi. During the tissue dissociation and staining for FACS sorting, all washes and antibody incubations were performed in Hank's Balanced Salt Solution without phenol red (Gibco) with 1% high grade BSA (Sigma A7979) and 0.1% glucose. All processing steps were performed on ice after the papain digestion. Cells were sorted into V-bottom 96-well plates (Corning, CLS3894) containing 150 μl DMEM/F12 (Gibco), with 1x insulin-transferrin-selenium (Gibco), 1x penicillin-streptomycin-glutamine (Gibco), 2% B-27 (Gibco), 2% N2 (Gibco), 15 mM Hepes, and 0.6% glucose (catching media). The plate and media were chilled to 4°C. All post-processing of cells was performed rapidly on ice immediately after sort completion until cells were lysed. One male and one female mouse were pooled to generate each of the 4 biological replicates that were FACS-sorted and sequenced for each age. Young and old sample pairs were FACS-sorted on the same day. To minimize batch effects from library preparation, all 40 libraries were made in the same batch. Four hundred cells of each cell type were input into an RNA-seq library. Poly-A selected, unstranded RNA-seq libraries were generated using the Clontech SMARTer Universal Low Input RNA Kit for cDNA production (634935) and the Nugen Ultralow DR Multiplex library kit for library generation (0330-32).
attributes.tag: ENA first public
attributes.value: 2018-10-08
attributes.tag: ENA last update
attributes.value: 2016-10-17
attributes.tag: External Id
attributes.value: SAMEA4515551
attributes.tag: INSDC center alias
attributes.value: Stanford University, Department of Genetics
attributes.tag: INSDC center name
attributes.value: Stanford University, Department of Genetics
attributes.tag: INSDC first public
attributes.value: 2018-10-08T17:02:50Z
attributes.tag: INSDC last update
attributes.value: 2016-10-17T14:56:45Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-5172:Ast_old_E
attributes.tag: age
attributes.value: 19 to 22
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: astrocyte
attributes.tag: common name
attributes.value: house mouse
attributes.tag: organism part
attributes.value: postnatal subventricular zone
attributes.tag: sample name
attributes.value: E-MTAB-5172:Ast_old_E
attributes.tag: sex
attributes.value: mixed