GSM7498810: Basolateral amygdala, snRNA-seq; Rattus norvegicus; RNA-Seq
For nuclei isolation, brain punches from 3 rats for each region were pooled and homogenized in homogenization buffer (0.26 M sucrose, 0.03 M KCl, 0.01 M MgCl2, 0.02 M Tricine-KOH pH 7.8, 0.001 M DTT, 0.5 mM spermidine, 0.15 mM Spermine, 0.3% NP40) using with 1ml glass Dounce homogenizers. The homogenate was filtered with a 70-um strainer filter (SP Bel-Art, cat no 136800070) and centrifuged for 5 minutes at 350 RCF. The nuclei were purified with an iodixanol gradient (Sigma-Aldrich # 92339-11-2) by layering a 25% Iodixanol-nuclei mixture on top of 30% and 40% Iodixanol solutions. After centrifugation at 4oC 3,000 RCF for 20 minutes, nuclei were collected from the 30-40% interface. Nuclei were washed in ATAC-RSB-Tween buffer (0.01 M Tris-HCl pH 7.5, 0.01 M NaCl, 0.003 M MgCl2, 0.1% Tween-20) and then resuspended in 1X nuclei buffer (10X genomics, PN 2000207). snRNA-seq library was performed using the Chromium Next GEM Single Cell Multiome Reagent Kit A (# 1000282) following Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kits User Guide (10X Genomics). After the transposition reaction, nuclei were encapsulated and barcoded. Next-generation sequencing libraries were constructed following the User Guide. 10X Genomics Multiome ATAC + Gene Expression