GSM4815731: P4_BNST_Female_Vehicle_3; Mus musculus; RNA-Seq
Four days after injection, animals were rapidly                     decapitated, and 400-µm sections were collected in ice-cold homogenization                     buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 120 mM tricine-KOH, pH 7.8) on a                     microtome. The BNST was microdissected (4 animals pooled per condition) and                     collected in 1 ml of cold homogenization buffer containing 1 mM DTT, 0.15 mM                     spermine, 0.5 mM spermidine, and 1X EDTA-free PIC, and 0.4 U/ml RNAseOUT. The                     tissue was dounce-homogenized 15x in a 1 ml glass tissue grinder (Wheaton) with                     a loose pestle. 0.3% IGEPAL CA-630 was added, and the suspension was homogenized                     5x with a tight pestle. The homogenate was filtered through a 40-µm strainer                     then centrifuged at 500 x g for 15 min at 4oC. The pellet was resuspended in 0.5                     ml homogenization buffer containing 1 mM DTT, 0.15 mM spermine, 0.5 mM                     spermidine, 1X EDTA-free PIC, and 0.2 U/ml RNAseOUT. 12,000 GFP+ nuclei were                     collected into cold Buffer RLT Plus supplemented 1:100 with ß-mercaptoethanol                     using the Sony SH800S Cell Sorter (purity mode) with a 100-µm sorting chip.                     Samples were stored at -80degC until RNA extraction. RNA extraction was                     performed with Qiagen RNeasy Plus Micro Kit. Libraries were prepared using the                     Tecan Genomics Ovation SoLo RNA-seq kit