GSM5917220: snRNA_hippocampus_VEH-CON_rep1; Mus musculus; RNA-Seq
To collect nuclei, frozen brain tissue was homogenized in a douncer filled with 6ml Solution D (0.25M Sucrose, 25mM KCl, 5mM MgCl2, 20mM Tris-HCl, pH 7.5, 5µg/mL actinomycin D (Sigma, Cat# A4262)). Samples were centrifuged for 1 min at max speed and pellets were resuspended in 4ml Solution D and 2ml Optiprep (Serumwerk Bernburg). Samples were pelleted by centrifugation for 10 min at 3,200g, and the supernatant was discarded. Optiprep purification was performed 2 times. After final centrifugation, pellets were resuspended in N-PBS (PBS, 5% BSA, 5µg/mL actinomycinD and 0.2U/µl RNAse inhibitor (Thermo Fisher Scientific, Cat#N8080119)) and filtered into 5ml polysterene tubes with filter (75mm) snap-caps (Corning, Cat# 352235). Library constructions were performed using Chromium SingleCell 3'Reagent Kit v3 chemistry (10x Genomics) according to the manufacturers protocol (CG000183 - Rev A). All 4 libraries were pooled and sequenced across 2 NextSeq 500 (v2.5) chips for 75 cycles. FASTQ files were generated using cellranger mkfastq (CellRanger v3.0.1), yielding an R1 length of 28nt and an R2 length of 56 nucleotides and one information file (I). single nuclear RNA-seq (10x)