GSM8720156: Control, Rep2, 3m, prefrontal cortex; Mus musculus; RNA-Seq
Frontal cortex of FXTAS mice and littermate controls was rapidly dissected in ice-cold homogenization buffer (HB: 260 mM sucrose, 30 mM KCl, 10 mM NaCl, 20 mM Tricine-KOH pH 7.8, 1 mM DTT, 0.5 mM Spermidine, 0.2 mM Spermine, 0.3% NP40, Complete Protease inhibitor (Roche), and Ribolock RNase inhibitor (ThermoFisher)) and immediately transferred to a Dounce homogenizer filled with 1 mL HB. Tissue was homogenized by 20 strokes with each pestle A and pestle B. The homogenate was then filtered through a 70um strainer followed by centrifugation for 10 min at 300g at 4°C. The pallets were then resuspended in a homogenization buffer and mixed with 1 volume of 50% Iodixanol (Sigma D1556) to make a final concentration of 25% iodixanol. Without disturbing the mixture, 30% Iodixanol was slowly layered underneath the 25% iodixanol mixture, and then 40% Iodixanol was gently added under the 30% mixture to generate a density gradient. A clearly defined interface should be visible between all three layers. The nuclei were centrifuged for 20 min at 1485g and 4°C in a swinging bucket centrifuge with the brake off. After the centrifugation, the nuclei band was transferred from the interface between 30% and 40% iodixanol solutions to a pre-chilled tube. The nuclei were washed with wash buffer at 4°C and counted after adding trypan blue. Libraries were generated with the 10x Genomics Single Cell Gene Expression reagent kit following the manufacturer's instructions with minor modifications. Briefly, per sample, 16,100 nuclei were resuspended in 1x diluted nuclei buffer (10x Genomics) with 2% BSA (Sigma), and 10,000 nuclei were captured. For 10,000 nuclei capture targeted, single nuclei expression libraries were sequenced to 20,000 read pairs per nuclei on an Illumina NovaSeq 6000 instrument.