The number of reads mapping to each gene (including introns) was calculated using featureCounts.ĭifferential expression analysis was performed with DESeq2. For the neonatal dataset, technical duplicate reads were removed using the manufacturer's nudup.py script ( ). The number of reads mapped to each gene (exons only) was calculated using featureCounts. For neonatal dataset, reads were adapter trimmed and quality filtered (q>30) using cutadapt.įiltered reads were mapped to the mm10 reference genome using STAR. Libraries were prepared using the Tecan Genomics Ovation SoLo RNA-seq kitįor adult dataset, reads were adapter trimmed and quality filtered (q>30) using the Fast-X toolkit ( ). RNA extraction was performed with Qiagen RNeasy Plus Micro Kit. Samples were stored at -80degC until RNA extraction. 12,000 GFP+ nuclei were collected into cold Buffer RLT Plus supplemented 1:100 with β-mercaptoethanol using the Sony SH800S Cell Sorter (purity mode) with a 100-μm sorting chip. 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. The homogenate was filtered through a 40-μm strainer then centrifuged at 500 x g for 15 min at 4oC.

0.3% IGEPAL CA-630 was added, and the suspension was homogenized 5x with a tight pestle. The tissue was dounce-homogenized 15x in a 1 ml glass tissue grinder (Wheaton) with a loose pestle. 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. Genotype: Esr1Cre/+ ROSA26CAG-Sun1-sfGFP-Myc/+Īnimals were injected subcutaneously with 5 ug estradiol benzoate or vehicle control (corn oil) on the day of birth (P0)Įsr1Cre/+ ROSA26CAG-Sun1-sfGFP-Myc/+ female miceįour 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. GEO help: Mouse over screen elements for information.

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