GSM8575017: SynCRE_E4_5M_2; Mus musculus; RNA-Seq
The mouse hippocampus was dissected on ice and placed into a pre-chilled 2 mL Dounce with 1 mL of cold 1X Homogenization Buffer (1X HB) (250 mM Sucrose, 25 mM KCL, 5 mM MgCl2, 20 mM Tricine-KOH pH7.8, 1 mM DTT, 0.5 mM Sermidine, 0.15 mM Sermine, 0.3% NP40, 0.2 units/µL RNase inhibitor, ~0.07 tablets/sample Protease inhibitor). Dounce with “A” loose pestle (~10 strokes) and then with “B” tight pestle (~15 strokes). The homogenate was filtered using a 70 µM Flowmi strainer (Eppendorf) and transferred to a pre-chilled 2 mL LoBind tube (Fischer Scientific). Nuclei were pelleted by spinning for 5 min at 4°C at 350 RCF. The supernatant was removed and the nuclei were resuspended in 400 µL 1X HB. Next, 400 µL of 50% Iodixanol solution was added to the nuclei and then slowly layered with 600 µL of 30% Iodixanol solution under the 25% mixture, then layered with 600 µL of 40% Iodixanol solution under the 30% mixture. The nuclei were then spun for 20 min at 4°C at 3,000g in a pre-chilled swinging bucket centrifuge. 200 µL of the nuclei band at the 30%-40% interface was collected and transferred to a fresh tube. Then, 800 µL of 2.5% BSA in PBS plus 0.2 units/µL of RNase inhibitor was added to the nuclei and then were spun for 10 min at 500 RCF at 4C. The nuclei were resuspended with 2% BSA in PBS plus 0.2 units/µL RNase inhibitor to reach ~500 nuclei/µL. The nuclei were then filtered with a 40 µM Flowmi stainer. The nuclei were counted and then ~13,000 nuclei per sample were loaded onto 10x Genomics Next GEM chip G. The snRNA-seq libraries were prepared using the Chromium Next GEM Single Cell 3ʹ Library and Gel Bead kit v3.1 (10x Genomics) according to the manufacturer's instructions. Libraries were sequenced on an Illumina NovaSeq 6000 sequencer at the UCSF CAT Core. The snRNA-seq libraries were prepared using the Chromium Next GEM Single Cell 3ʹ Library and Gel Bead kit v3.1 (10x Genomics) according to the manufacturer's instructions. Libraries were sequenced on an Illumina NovaSeq 6000 sequencer at the UCSF CAT Core. 10X Single-nucleus RNAseq