Dissociated cells were layered on top of 10ml of high concentration inhibitor solution with 5mg/ml BSA and 5mg/ml ovomucoid and centrifuged at 130g for 5 minutes. Louis, MO, A-8806), and ovomucoid (also known as trypsin inhibitor, 1.0 mg/ml, Roche Diagnostics Corporation, Indianapolis, IN 109878) and then mechanically dissociated by gentle sequential trituration using a 5ml pipette. Following papain treatment, the tissue was washed three times with 4.5ml of inhibitor buffer containing BSA (1.0mg/ml, Sigma, St. The papain solution was equilibrated with 5% CO2 and 95% O2 gas before and during papain treatment. Briefly, the tissue was incubated at 33 ☌ for 45 minutes in 20 ml of a papain solution containing Earle’s balanced salts (EBSS, Sigma, St. The tissue was enzymatically dissociated to make a suspension of single cells as described previously. The cortices were dissected out and meninges were removed. Cells from a litter of 8-16 P7 Aldh1l1-EGFP transgenic mice of both genders were pooled together as one biological replicate. This line has been previously characterized to have complete astrocyte-specific labeling throughout the brain. Method Part1:To purify astrocytes, we took advantage of a BAC transgenic mouse line expressing EGFP under the control of regulatory sequences in Aldh1l1-BAC.
Our analysis identified thousands of new cell type enriched genes and splicing isoforms that will provide novel markers for cell identification, new tools for genetic manipulation, and numerous insights into the biology of the brain.
We generated a transcriptome database for these 8 cell types by RNA sequencing and used a highly sensitive algorithm to detect alternative splicing events in each gene.
Purpose: To better understand the function of the various cell types of the brain, we prospectively purified neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. GEO help: Mouse over screen elements for information.Īn RNA-Seq transcriptome and splicing database of neurons, glia, and vascular cells of the cerebral cortexĮxpression profiling by high throughput sequencing
0 kommentar(er)
