GSM6257079: Ketogenic Diet Astrocytes - R4; Mus musculus; RNA-Seq
Cells were isolated from cortex according to the adult brain dissociation protocol (Miltenyi Biotec). Antibody labeling was done according to the Microbead kit protocols using the antibodies specific to markers for the following cell types: oligodendrocytes (O4: 1:40; 130-096-670), astrocytes (ACSA-2: 1:10; 130-097-679), microglia (CD11b: 1:10; 130-093-636) and endothelial cells (CD31: 1:10; 130-091-935). Neurons were isolated by negative selection. The purity of cell populations was routinely determined by qPCR on extracted and reverse-transcribed RNA. RNA was extracted with the Qiagen Micro-RNeasy Kit (Qiagen, Cat. No. 74004) according to the manufacturer's protocol. As external control, 1µl of ERCC RNA Spike-In Mix (ThermoFisher, Cat No. 4456740) diluted at 1:5000 was added to the isolated RNA. cDNA was synthesised with the Ovation RNA-Seq system v2 (NuGEN, Cat. No.7102). As input, 100ng of cDNA was used for library preparation using the IonXpress plus gDNA and Amplicon library preparation kit (Thermofischer,cat no: 4471269). To achieve size selection of the library, a 2% E-Gel was used and the samples were barcoded and subsequently amplified. Further quantification for each sample library was performed using the Kapa Library Quantification Kit (Kapa, Cat. No. KK4827). Of each sample, equal amounts were sequenced on an Ion Torrent Proton Sequencer.