Dataset Information

An RNA-Seq transcriptome and splicing database of neurons, glia, and vascular cells of the cerebral cortex

ABSTRACT: 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. 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. 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. 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. This line has been previously characterized to have complete astrocyte-specific labeling throughout the brain. Cells from a litter of 8-16 P7 Aldh1l1-EGFP transgenic mice of both genders were pooled together as one biological replicate. The cortices were dissected out and meninges were removed. The tissue was enzymatically dissociated to make a suspension of single cells as described previously. Briefly, the tissue was incubated at 33 °C for 45 minutes in 20 ml of a papain solution containing Earle’s balanced salts (EBSS, Sigma, St. Louis, MO, E7510), D(+)-glucose (22.5mM), NaHCO3 (26mM), DNase (125U/ml, Worthington, Lakewood, NJ, LS002007), papain (9 U/ml, Worthington, Lakewood, NJ, LS03126), and L-cysteine (1mM, Sigma, St. Louis, MO, C7880). The papain solution was equilibrated with 5% CO2 and 95% O2 gas before and during papain treatment. Following papain treatment, the tissue was washed three times with 4.5ml of inhibitor buffer containing BSA (1.0mg/ml, Sigma, St. 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. 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. The cell pellet was then resuspended in 12 ml Dulbecco’s phosphate-buffered saline (DPBS, Invitrogen, Carlsbad, CA 14287) containing 0.02% BSA and 12.5U/ml DNase and filtered through a 20um Nitex mesh (Sefar America Inc., Depew NY, Lab Pak 03-20/14) to remove undissociated cell clumps. This yields a single cell suspension. Cell health is assessed by trypan blue exclusion. Only single cell suspensions with >85% viability were used for purification experiments. 1μg/ml propidium iodide (PI, Sigma, St. Louis, MO, P4864) was added to the single cell solution to label dead cells. Cells were sorted on a BD Aria II cell sorter (BD Bioscience) with a 70μm nozzle. Dead cells and debris were gated first by their low forward light scatter and high side light scatter and secondly by high PI staining. Doublets were removed by high side light scatter. Cell concentration and flow rate were carefully adjusted to maximize purity. Astrocytes were identified based on high EGFP fluorescence. FACS routinely yielded >99% purity based on reanalysis of sorted cells. Method Part2:To purify neurons, a single cell suspension was prepared as described above and incubated at 34 °C for one hour to allow expression of cell surface protein antigens digested by papain, and then incubated on two sequential panning plates coated with BSL-1 to deplete endothelial cells (10 minutes each), followed by a 30 minute incuation on a plate coated with mouse IgM anti-O4 hybridoma (Bansal et al., 1989. 4ml hybridoma supernatant diluted with 8ml DPBS/0.2% BSA) to deplete OPCs, and then incubated for 20 minutes on a plate coated with rat anti-mouse CD45 (BD Pharmingen 550539, 1.25ug in 12ml of DPBS/0.2% BSA) to deplete microglia and macrophages. Finally cells were added to a plate coated with rat anti-mouse L1CAM (30ug in 12ml of DPBS/0.2% BSA, Millipore, Billerica, MA, MAB5272) to bind neurons. The adherent cells on the L1CAM plate were washed 8 times with 10-20 ml of DPBS to remove all antigen-negative nonadherent cells, and then removed from the plate by treating with trypsin (Sigma, 1,000U/ml, T-4665) in 8ml Ca2+ and Mg2+ free EBSS (Irvine Scientific, Santa Ana, CA, 9208) for 3-10 minutes at 37°C in a 10% CO2 incubator. The trypsin was then neutralized with 20ml of fetal calf serum (FCS) solution containing 30% FCS (Gibco, 10437-028), 35% Dulbecco’s modified eagle medium (DMEM, Invitrogen, 11960-044), and 35% Neurobasal (Gibco, 21103-049). The cells were dislodged by gentle squirting of FCS solution over the plate and harvested by centrifugation at 200g for 10 minutes. Method Part3:To purify microglia and oligodendrocyte-lineage cells, the mice were first perfused with 10ml PBS to remove macrophage contamination from the brain. A single cell suspension was then prepared as described above and incubated 20 minutes on a plate coated with rat anti-mouse CD45 (BD Pharmingen 550539, 1.25ug in 12ml of DPBS/0.2% BSA) to harvest microglia, and then incubated sequentially on four BSL1 coated plates (8 minutes each) to deplete endothelial cells and remaining microglia. The remaining cells were next incubated for 30 minutes on a rat anti-PDGFRα (10ug in 12ml DPBS/0.2% BSA, Fitzgerald, Acton, MA, 10R-CD140aMS) coated plate to harvest OPCs, and then incubated on an additional PDGFRα plate and mouse A2B5 monoclonal antibody ascites (American Type Culture Collection, Rockville, MD) coated plate for 30 minutes each to deplete remaining OPCs. The cell suspension was next incubated on an anti-MOG hybridoma coated plate for 30 minutes to harvest myelinating oligodendrocytes, followed by an additional anti-MOG hybridoma coated plate for 30 minutes to deplete any remaining myelinating oligodendrocytes. Finally, the cell suspension was incubated on an anti-GalC hybridoma coated plate for 30 minutes to harvest newly formed oligodendrocytes. For purification of RNA, the cells were lysed while still attached to the panning plate with Qiazol reagent (Qiagen 217004), and total RNA was purified as described below. Mehtod Part4:To purify endothelial cells, we took advantage of Tie2-EGFP transgenic mice available from Jackson labs. These mice express EGFP under the pan-endothelial Tie2 promotor (Daneman et al., 2010; Motoike et al., 2000). Single cell suspension was prepared and FACS was performed as described above. Method Part5: RNA-Seq was performed on the polyadenylated fraction of RNA isolated from purified cell samples.Two biological replicates were used for each phase.100 ng total RNAs were used for each sequencing library. RNA samples were polyA selected and paired-end sequencing libraries were constructed using TruSeq RNA Sample Prep Kit as described in the TruSeq RNA Sample Preparation V2 Guide (Illumina).The samples were then sequenced using the Illumina HiSeq 2000 sequencer. Method Part6: Read mapping and Transcriptome construction were done by using optimized pipeline which integrate Tophat followed by Cufflinks. Result:We purified neurons, astrocytes, oligodendrocyte precursor cells (OPCs), newly formed oligodendrocytes (NFOs), myelinating oligodendrocytes (MOs), microglia, endothelial cells and pericytes from mouse cortex and used RNA-Seq to generate a high resolution transcriptome database of > 22,000 genes. We identified thousands of novel cell type-enriched genes that have not been previously identified. These include novel transcription factors, ligands, receptors, enzymes, and signaling molecules. We then used a novel splice mapping algorithm to identify thousands of cell type-specific alternative splicing events. Conclusion: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. Our analysis i mRNA profiles of purified cell samples from mice were generated by RNA-sequencing, in duplicate, using Illumina HiSeq 2000.

REANALYSIS of: E-GEOD-52564

ORGANISM(S): Mus musculus

PROVIDER: E-GEOD-52564 | ExpressionAtlas | 2015-07-29

REPOSITORIES: ExpressionAtlas

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An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex.

Zhang Ye Y Chen Kenian K Sloan Steven A SA Bennett Mariko L ML Scholze Anja R AR O'Keeffe Sean S Phatnani Hemali P HP Guarnieri Paolo P Caneda Christine C Ruderisch Nadine N Deng Shuyun S Liddelow Shane A SA Zhang Chaolin C Daneman Richard R Maniatis Tom T Barres Ben A BA Wu Jian Qian JQ

The Journal of Neuroscience 20140901 36

The major cell classes of the brain differ in their developmental processes, metabolism, signaling, and function. To better understand the functions and interactions of the cell types that comprise these classes, we acutely purified representative populations of neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. We generated a transcriptome database for these ei ...[more]

PMID: 25186741

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Project description: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. 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. 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. 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. This line has been previously characterized to have complete astrocyte-specific labeling throughout the brain. Cells from a litter of 8-16 P7 Aldh1l1-EGFP transgenic mice of both genders were pooled together as one biological replicate. The cortices were dissected out and meninges were removed. The tissue was enzymatically dissociated to make a suspension of single cells as described previously. Briefly, the tissue was incubated at 33 °C for 45 minutes in 20 ml of a papain solution containing Earle’s balanced salts (EBSS, Sigma, St. Louis, MO, E7510), D(+)-glucose (22.5mM), NaHCO3 (26mM), DNase (125U/ml, Worthington, Lakewood, NJ, LS002007), papain (9 U/ml, Worthington, Lakewood, NJ, LS03126), and L-cysteine (1mM, Sigma, St. Louis, MO, C7880). The papain solution was equilibrated with 5% CO2 and 95% O2 gas before and during papain treatment. Following papain treatment, the tissue was washed three times with 4.5ml of inhibitor buffer containing BSA (1.0mg/ml, Sigma, St. 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. 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. The cell pellet was then resuspended in 12 ml Dulbecco’s phosphate-buffered saline (DPBS, Invitrogen, Carlsbad, CA 14287) containing 0.02% BSA and 12.5U/ml DNase and filtered through a 20um Nitex mesh (Sefar America Inc., Depew NY, Lab Pak 03-20/14) to remove undissociated cell clumps. This yields a single cell suspension. Cell health is assessed by trypan blue exclusion. Only single cell suspensions with >85% viability were used for purification experiments. 1μg/ml propidium iodide (PI, Sigma, St. Louis, MO, P4864) was added to the single cell solution to label dead cells. Cells were sorted on a BD Aria II cell sorter (BD Bioscience) with a 70μm nozzle. Dead cells and debris were gated first by their low forward light scatter and high side light scatter and secondly by high PI staining. Doublets were removed by high side light scatter. Cell concentration and flow rate were carefully adjusted to maximize purity. Astrocytes were identified based on high EGFP fluorescence. FACS routinely yielded >99% purity based on reanalysis of sorted cells. Method Part2:To purify neurons, a single cell suspension was prepared as described above and incubated at 34 °C for one hour to allow expression of cell surface protein antigens digested by papain, and then incubated on two sequential panning plates coated with BSL-1 to deplete endothelial cells (10 minutes each), followed by a 30 minute incuation on a plate coated with mouse IgM anti-O4 hybridoma (Bansal et al., 1989. 4ml hybridoma supernatant diluted with 8ml DPBS/0.2% BSA) to deplete OPCs, and then incubated for 20 minutes on a plate coated with rat anti-mouse CD45 (BD Pharmingen 550539, 1.25ug in 12ml of DPBS/0.2% BSA) to deplete microglia and macrophages. Finally cells were added to a plate coated with rat anti-mouse L1CAM (30ug in 12ml of DPBS/0.2% BSA, Millipore, Billerica, MA, MAB5272) to bind neurons. The adherent cells on the L1CAM plate were washed 8 times with 10-20 ml of DPBS to remove all antigen-negative nonadherent cells, and then removed from the plate by treating with trypsin (Sigma, 1,000U/ml, T-4665) in 8ml Ca2+ and Mg2+ free EBSS (Irvine Scientific, Santa Ana, CA, 9208) for 3-10 minutes at 37°C in a 10% CO2 incubator. The trypsin was then neutralized with 20ml of fetal calf serum (FCS) solution containing 30% FCS (Gibco, 10437-028), 35% Dulbecco’s modified eagle medium (DMEM, Invitrogen, 11960-044), and 35% Neurobasal (Gibco, 21103-049). The cells were dislodged by gentle squirting of FCS solution over the plate and harvested by centrifugation at 200g for 10 minutes. Method Part3:To purify microglia and oligodendrocyte-lineage cells, the mice were first perfused with 10ml PBS to remove macrophage contamination from the brain. A single cell suspension was then prepared as described above and incubated 20 minutes on a plate coated with rat anti-mouse CD45 (BD Pharmingen 550539, 1.25ug in 12ml of DPBS/0.2% BSA) to harvest microglia, and then incubated sequentially on four BSL1 coated plates (8 minutes each) to deplete endothelial cells and remaining microglia. The remaining cells were next incubated for 30 minutes on a rat anti-PDGFRα (10ug in 12ml DPBS/0.2% BSA, Fitzgerald, Acton, MA, 10R-CD140aMS) coated plate to harvest OPCs, and then incubated on an additional PDGFRα plate and mouse A2B5 monoclonal antibody ascites (American Type Culture Collection, Rockville, MD) coated plate for 30 minutes each to deplete remaining OPCs. The cell suspension was next incubated on an anti-MOG hybridoma coated plate for 30 minutes to harvest myelinating oligodendrocytes, followed by an additional anti-MOG hybridoma coated plate for 30 minutes to deplete any remaining myelinating oligodendrocytes. Finally, the cell suspension was incubated on an anti-GalC hybridoma coated plate for 30 minutes to harvest newly formed oligodendrocytes. For purification of RNA, the cells were lysed while still attached to the panning plate with Qiazol reagent (Qiagen 217004), and total RNA was purified as described below. Mehtod Part4:To purify endothelial cells, we took advantage of Tie2-EGFP transgenic mice available from Jackson labs. These mice express EGFP under the pan-endothelial Tie2 promotor (Daneman et al., 2010; Motoike et al., 2000). Single cell suspension was prepared and FACS was performed as described above. Method Part5: RNA-Seq was performed on the polyadenylated fraction of RNA isolated from purified cell samples.Two biological replicates were used for each phase.100 ng total RNAs were used for each sequencing library. RNA samples were polyA selected and paired-end sequencing libraries were constructed using TruSeq RNA Sample Prep Kit as described in the TruSeq RNA Sample Preparation V2 Guide (Illumina).The samples were then sequenced using the Illumina HiSeq 2000 sequencer. Method Part6: Read mapping and Transcriptome construction were done by using optimized pipeline which integrate Tophat followed by Cufflinks. Result:We purified neurons, astrocytes, oligodendrocyte precursor cells (OPCs), newly formed oligodendrocytes (NFOs), myelinating oligodendrocytes (MOs), microglia, endothelial cells and pericytes from mouse cortex and used RNA-Seq to generate a high resolution transcriptome database of > 22,000 genes. We identified thousands of novel cell type-enriched genes that have not been previously identified. These include novel transcription factors, ligands, receptors, enzymes, and signaling molecules. We then used a novel splice mapping algorithm to identify thousands of cell type-specific alternative splicing events. Conclusion: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. Our analysis i mRNA profiles of purified cell samples from mice were generated by RNA-sequencing, in duplicate, using Illumina HiSeq 2000.

Project description:A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function Understanding the cell-cell interactions that control CNS development and function has long been limited by the lack of methods to cleanly separate astrocytes, neurons, and oligodendrocytes. Here we describe the first method for the isolation and purification of developing and mature astrocytes from mouse forebrain. This method takes advantage of the expression of S100β by astrocytes. We used fluorescent activated cell sorting (FACS) to isolate EGFP positive cells from transgenic mice that express EGFP under the control of an S100β promoter. By depletion of astrocytes and oligodendrocytes we obtained purified populations of neurons, while by panning with oligodendrocyte-specific antibodies we obtained purified populations of oligodendrocytes. Using GeneChip Arrays we then created a transcriptome database of the expression levels of over 20,000 genes by gene profiling these three main CNS neural cell types at postnatal ages day 1 to 30. This database provides the first global characterization of the genes expressed by mammalian astrocytes in vivo and is the first direct comparison between the astrocyte, neuron, and oligodendrocyte transcriptomes. We demonstrate that Aldh1L1, a highly expressed astrocyte gene, is a highly specific antigenic marker for astrocytes with a substantially broader, and therefore potentially more useful, pattern of astrocyte expression than the traditional astrocyte marker GFAP. This transcriptome database of acutely isolated and highly pure populations of astrocytes, neurons and oligodendrocytes provides a resource to the neuroscience community by providing improved cell type specific markers and for better understanding of neural development, function, and disease. We acutely purified mouse astrocytes from early postnatal ages (P1) to later postnatal ages (P30), when astrocyte differentiation is morphologically complete (Bushong et al., 2004), and acutely purified mouse OL-lineage cells from stages ranging from OPCs to newly differentiated OLs to myelinating OLs. We extracted RNA from each of these highly purified, acutely isolated cell types and used GeneChip Arrays to determine the expression levels of over 20,000 genes and construct a comprehensive database of cell type specific gene expression in the mouse forebrain. Analysis of this database confirms cell type specific expression of many well characterized and functionally important genes. In addition, we have identified thousands of new cell type enriched genes, thereby providing important new information about astrocyte, OL, and neuron interactions, metabolism, development, and function. This database provides a comparison of the genome-wide transcriptional profiles of the main CNS cell types and is a resource to the neuroscience community for better understanding the development, physiology, and pathology of the CNS. Keywords: Developmental CNS Cell type comparision Overall design: FACS purification of astrocytes: Dissociated forebrains from S100β-EGFP mice were resuspended in panning buffer (DBPS containing 0.02% BSA and 12.5 U/ml DNase) and sequentially incubated on the following panning plates: secondary antibody only plate to deplete microglia, O4 plate to deplete OLs, PDGFRα plate to deplete OPCs, and a second O4 plate to deplete any remaining OLs. This procedure was sufficient to deplete all OL-lineage cells from animals P8 and younger, however, in older animals that had begun to myelinate, additional depletion of OLs and myelin debris was accomplished as follows. The nonadherent cells from the last O4 dish were harvested by centrifugation, and the cells were resuspended in panning buffer containing GalC, MOG, and O1 supernatant and incubated for 15 minutes at room temperature. The cell suspension was washed and then resuspended in panning buffer containing 20 μg donkey anti-mouse APC for 15 minutes. The cells were washed and resuspended in panning buffer containing propidium iodide (PI). EGFP+ astrocytes were then purified by fluorescence activated cell sorting (FACS). Dead cells were gated out using high PI staining and forward light scatter. Astrocytes were identified based on high EGFP fluorescence and negative APC fluorescence from indirect immunostaining for OL markers GalC, MOG, and O1. Cells were sorted twice and routinely yielded >99.5% purity based on reanalysis of double sorted cells.; FACS purification of neurons: EGFP- cells were the remaining forebrain cells after microglia, OLs, and astrocytes had been removed, and were primarily composed of neurons, and to a lesser extent, endothelial cells (we estimate < 4% endothelial cells at P7 and < 20% endothelial cells at P17). EGFP- cells from S100β-EGFP dissociated forebrain were FACS purified in parallel with astrocyte purification and were sorted based on their negative EGFP fluorescence immunofluorescence. Cells were sorted twice and routinely yielded >99.9% purity. In independent preparations, the EGFP- cell population was additionally depleted of endothelial cells and pericytes by sequentially labeling with biotin-BSL1 lectin and streptavidin-APC while also labeling for OL markers as described above. Cells were sorted twice and routinely yielded >99.9% purity.; Panning purification of oligodendrocyte lineage cells: Dissociated mouse forebrains were resuspended in panning buffer. In order to deplete microglia, the single-cell suspension was sequentially panned on four BSL1 panning plates. The cell suspension was then sequentially incubated on two PDGFRα plates (to purify and deplete OPCs), one A2B5 plate (to deplete any remaining OPCs), two MOG plates (to purify and deplete myelinating OLs), and one GalC plate (to purify the remaining PDGFRα-, MOG-, OLs). The adherent cells on the first PDGFRα, MOG, and GalC plates were washed to remove all antigen-negative nonadherent cells. The cells were then lysed while still attached to the panning plate with Qiagen RLT lysis buffer, and total RNA was purified. Purified OPCs were >95% NG2 positive and 0% MOG positive. Purified Myelin OLs were 100% MOG positive, >95% MBP positive, and 0% NG2 positive. Purified GalC OLs depleted of OPCs and Myelin OLs were <10% MOG positive and ~50% weakly NG2 positive, a reflection of their recent development as early OLs.; Data normalization and analysis: Raw image files were processed using Affymetrix GCOS and the MAS 5.0 algorithm. Intensity data was normalized per chip to a target intensity TGT value of 500, and expression data and absent/present calls for individual probe sets were determined. Gene expression values were normalized and modeled across arrays using the dChip software package with invariant-set normalization and a PM model. (www.dchip.org, Li and Wong, 2001). The 29 samples were grouped into 9 sample types: Astros P7-P8, Astros P17, Astros P17-gray matter (P17g), Neurons P7, Neurons P17, Neurons-endothelial cell depleted (P7n, P17n), OPCs, GalC-OLs, and MOG-OLs. Gene filtering was performed to select probe sets that were consistently expressed in at least one cell type, where consistently expressed was defined as being called present and having a MAS 5.0 intensity level greater than 200 in at least two-thirds of the samples in the cell type. We identified 20,932 of the 45,037 probe sets that were consistently expressed in at least one of the nine cell types. The Significance Analysis of Microarrays (SAM) method (Tusher et al., 2001) was used to determine genes that were significantly differentially expressed between different cell types (see Supplemental Table S2 for SAM cell type groupings). Clustering was performed using the hclust method with complete linkage in R. Expression values were transformed for clustering by computing a mean expression value for the gene using those samples in the corresponding SAM statistical analysis, and then subtracting the mean from expression intensities. In order to preserve the log2 scale of the data, unless otherwise indicated, no normalization by variance was performed. Plots were created using the gplots package in R. The Bioconductor software package (Gentleman et al., 2004) was used throughout the expression analyses. Functional analyses were performed through the use of Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com).

Project description:A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function Understanding the cell-cell interactions that control CNS development and function has long been limited by the lack of methods to cleanly separate astrocytes, neurons, and oligodendrocytes. Here we describe the first method for the isolation and purification of developing and mature astrocytes from mouse forebrain. This method takes advantage of the expression of S100β by astrocytes. We used fluorescent activated cell sorting (FACS) to isolate EGFP positive cells from transgenic mice that express EGFP under the control of an S100β promoter. By depletion of astrocytes and oligodendrocytes we obtained purified populations of neurons, while by panning with oligodendrocyte-specific antibodies we obtained purified populations of oligodendrocytes. Using GeneChip Arrays we then created a transcriptome database of the expression levels of over 20,000 genes by gene profiling these three main CNS neural cell types at postnatal ages day 1 to 30. This database provides the first global characterization of the genes expressed by mammalian astrocytes in vivo and is the first direct comparison between the astrocyte, neuron, and oligodendrocyte transcriptomes. We demonstrate that Aldh1L1, a highly expressed astrocyte gene, is a highly specific antigenic marker for astrocytes with a substantially broader, and therefore potentially more useful, pattern of astrocyte expression than the traditional astrocyte marker GFAP. This transcriptome database of acutely isolated and highly pure populations of astrocytes, neurons and oligodendrocytes provides a resource to the neuroscience community by providing improved cell type specific markers and for better understanding of neural development, function, and disease. We acutely purified mouse astrocytes from early postnatal ages (P1) to later postnatal ages (P30), when astrocyte differentiation is morphologically complete (Bushong et al., 2004), and acutely purified mouse OL-lineage cells from stages ranging from OPCs to newly differentiated OLs to myelinating OLs. We extracted RNA from each of these highly purified, acutely isolated cell types and used GeneChip Arrays to determine the expression levels of over 20,000 genes and construct a comprehensive database of cell type specific gene expression in the mouse forebrain. Analysis of this database confirms cell type specific expression of many well characterized and functionally important genes. In addition, we have identified thousands of new cell type enriched genes, thereby providing important new information about astrocyte, OL, and neuron interactions, metabolism, development, and function. This database provides a comparison of the genome-wide transcriptional profiles of the main CNS cell types and is a resource to the neuroscience community for better understanding the development, physiology, and pathology of the CNS. Keywords: Developmental CNS Cell type comparision FACS purification of astrocytes: Dissociated forebrains from S100β-EGFP mice were resuspended in panning buffer (DBPS containing 0.02% BSA and 12.5 U/ml DNase) and sequentially incubated on the following panning plates: secondary antibody only plate to deplete microglia, O4 plate to deplete OLs, PDGFRα plate to deplete OPCs, and a second O4 plate to deplete any remaining OLs. This procedure was sufficient to deplete all OL-lineage cells from animals P8 and younger, however, in older animals that had begun to myelinate, additional depletion of OLs and myelin debris was accomplished as follows. The nonadherent cells from the last O4 dish were harvested by centrifugation, and the cells were resuspended in panning buffer containing GalC, MOG, and O1 supernatant and incubated for 15 minutes at room temperature. The cell suspension was washed and then resuspended in panning buffer containing 20 μg donkey anti-mouse APC for 15 minutes. The cells were washed and resuspended in panning buffer containing propidium iodide (PI). EGFP+ astrocytes were then purified by fluorescence activated cell sorting (FACS). Dead cells were gated out using high PI staining and forward light scatter. Astrocytes were identified based on high EGFP fluorescence and negative APC fluorescence from indirect immunostaining for OL markers GalC, MOG, and O1. Cells were sorted twice and routinely yielded >99.5% purity based on reanalysis of double sorted cells.; FACS purification of neurons: EGFP- cells were the remaining forebrain cells after microglia, OLs, and astrocytes had been removed, and were primarily composed of neurons, and to a lesser extent, endothelial cells (we estimate < 4% endothelial cells at P7 and < 20% endothelial cells at P17). EGFP- cells from S100β-EGFP dissociated forebrain were FACS purified in parallel with astrocyte purification and were sorted based on their negative EGFP fluorescence immunofluorescence. Cells were sorted twice and routinely yielded >99.9% purity. In independent preparations, the EGFP- cell population was additionally depleted of endothelial cells and pericytes by sequentially labeling with biotin-BSL1 lectin and streptavidin-APC while also labeling for OL markers as described above. Cells were sorted twice and routinely yielded >99.9% purity.; Panning purification of oligodendrocyte lineage cells: Dissociated mouse forebrains were resuspended in panning buffer. In order to deplete microglia, the single-cell suspension was sequentially panned on four BSL1 panning plates. The cell suspension was then sequentially incubated on two PDGFRα plates (to purify and deplete OPCs), one A2B5 plate (to deplete any remaining OPCs), two MOG plates (to purify and deplete myelinating OLs), and one GalC plate (to purify the remaining PDGFRα-, MOG-, OLs). The adherent cells on the first PDGFRα, MOG, and GalC plates were washed to remove all antigen-negative nonadherent cells. The cells were then lysed while still attached to the panning plate with Qiagen RLT lysis buffer, and total RNA was purified. Purified OPCs were >95% NG2 positive and 0% MOG positive. Purified Myelin OLs were 100% MOG positive, >95% MBP positive, and 0% NG2 positive. Purified GalC OLs depleted of OPCs and Myelin OLs were <10% MOG positive and ~50% weakly NG2 positive, a reflection of their recent development as early OLs.; Data normalization and analysis: Raw image files were processed using Affymetrix GCOS and the MAS 5.0 algorithm. Intensity data was normalized per chip to a target intensity TGT value of 500, and expression data and absent/present calls for individual probe sets were determined. Gene expression values were normalized and modeled across arrays using the dChip software package with invariant-set normalization and a PM model. (www.dchip.org, Li and Wong, 2001). The 29 samples were grouped into 9 sample types: Astros P7-P8, Astros P17, Astros P17-gray matter (P17g), Neurons P7, Neurons P17, Neurons-endothelial cell depleted (P7n, P17n), OPCs, GalC-OLs, and MOG-OLs. Gene filtering was performed to select probe sets that were consistently expressed in at least one cell type, where consistently expressed was defined as being called present and having a MAS 5.0 intensity level greater than 200 in at least two-thirds of the samples in the cell type. We identified 20,932 of the 45,037 probe sets that were consistently expressed in at least one of the nine cell types. The Significance Analysis of Microarrays (SAM) method (Tusher et al., 2001) was used to determine genes that were significantly differentially expressed between different cell types (see Supplemental Table S2 for SAM cell type groupings). Clustering was performed using the hclust method with complete linkage in R. Expression values were transformed for clustering by computing a mean expression value for the gene using those samples in the corresponding SAM statistical analysis, and then subtracting the mean from expression intensities. In order to preserve the log2 scale of the data, unless otherwise indicated, no normalization by variance was performed. Plots were created using the gplots package in R. The Bioconductor software package (Gentleman et al., 2004) was used throughout the expression analyses. Functional analyses were performed through the use of Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com).