Bulk RNASeq of mouse astrocytes isolated from the somatosensory cortex of control and Tcf7l2 conditional knockout (TdTomato+).
Bulk RNASeq of mouse astrocytes isolated from the somatosensory cortex of control and Tcf7l2 conditional knockout (TdTomato+).
Mice were perfused with artificial CSF artificial cerebrospinal fluid that was composed of NaCl (Chempur, catalog no. 794121116, 119 mM concentration), KCl (Chempur, catalog no. 117397402, 2.5 mM concentration), MgSO4 (Sigma-Aldrich, catalog no. 1.06067, 1.3 mM concentration), CaCl2 (Chempur, catalog no. PA-07-12335, 2.5 mM concentration), NaH2PO4 (Sigma-Aldrich, catalog no. 74092, 1 mM concentration), NaHCO3 (Merck Life Science, catalog no. S5761-500G, 26.2 mM concentration), and glucose (Merck Life Science, catalog no. G7021-100G, 11 mM concentration), equilibrated with 95%/5% O2/CO2. Somatosensory cortex from Control TdT and Tcf7l2 cKO TdT mice (P35) were dissected and dissociated with papain (Worthington Biochemical, catalog no. LS003126, 20 U/ml concentration) for 70 min at 34°C. Next, tissue was dissociated in Low Ovo Inhibitor Solution (EBSS [Earle′s Balanced Salt Solution], Merck Life Science, catalog no. E7510; glucose, Merck Life Science, catalog no. G7021-100G, 0.46% concentration; NaHCO3, Merck Life Science, catalog no. S5761-500G, 26 mM concentration; bovine serum albumin [BSA], Merck Life Science, catalog no. A8806, 1 mg/ml concentration; trypsin inhibitor, Merck Life Science, catalog no. 9253-1G, 1 mg/ml concentration) and centrifuged for 10 min at 1200 rotations per minute, diluted in 1% BSA in PBS. Cell sorting was performed using BD FACS Aria II (BD Biosciences). Enhanced GFP (eGFP)-positive or TdT-positive cells were collected in post-sorting buffer (0.5% BSA, 10 mM HEPES, and 0.5% glucose [Merck Life Science, catalog no. G7021-100G]) and centrifuged at 500 g g for 10 min. The supernatant was removed, and the pellet was frozen at -80C. RNA from sorted cells (approximately 200,000 cells per sample) was isolated using the RNeasy Micro Kit (Qiagen, catalog no. 74004, TdT+ cells) and the quality of RNA was verified using Bioanalyzer (Agilent) Libraries were constructed accordingly to the protocol of Lexogen Quant Seq 3'mRNA Seq - Catalog Number 015 QuantSeq 3' mRNA-Seq Library Prep Kit for Illumina (FWD)