Form and function of actin impacts actin health and aging - snRNAseq
Form and function of actin impacts actin health and aging - snRNAseq
Worms were synchronized using a bleach-based egg isolation method and starved in M9 buffer for 24 h at the L1 stage. Following L1 arrest, worms were transferred to RNAi plates seeded with the appropriate bacteria and grown at 23 °C for 72 h to reach the day 1 adult stage. Approximately 3,000 day 1 adults per condition were then collected by washing the plates with M9 buffer. All buffers were made fresh prior to worm collection and placed on ice. All tips, tubes, and pestles were rinsed with homogenization buffer or PBS with BSA to reduced adhesion of nuclei to the surfaces. Collected worms were washed 3x with M9 in a 15mL tube and then transferred to a 1.5mL tube. The tubes were spun down in a swinging bucket centrifuge at 4C and the supernatant was removed before the samples were placed on ice. 100uL of homogenization buffer was added to the worm and then they were ground with a motorized pestle for 45 seconds. 800uL of homogenization buffer was added to the samples while used to wash the pestle. The samples were transferred to sterile 1-ml Dounce homogenizers on ice (Wheaton, 357538). 20 strokes were applied to the samples using the loose pestle and 50uL of homogenization buffer were used to rinse the pestle into the sample. 20 strokes of the tight pestle were then applied and 50uL of homogenization buffer were once again used to rinse the pestle into the sample. The lysates were then filtered into a 35uM cell strainer tube, followed by a Flowmi cell strainer (Bel-Art, H13680-0040) to transfer the lysate to a 1.5mL tube. The filtered lysates were spun at 800xG for 10 minutes at 4C using a swing bucket rotor. The supernatant was removed and the pellet was resuspended in 500uL of PBS with 0.5% BSA and RNAase inhibitor by pipetting 60 times and avoiding the formation of bubbles. Once the pellet was fully resuspended, the samples were filtered once more through a Flowmi cell strainer while being transferred to a flow cytometry tube. 20uL of the filtered samples was transferred into a tube containing 180uL of PBS with 0.5% BSA and RNAase inhibitor to be used as an unstained control for Fluorescence Activated Cell Sorting (FACS). The remaining sample was stained on ice using Hoechst (Invitrogen, 33342) at a working concentration of 1:1,000. Approximately 200,000 to 300,000 Hoechst positive events were sorted based on forward scatter area (FSC-A) into a 1.5mL tube. Sorted nuclei were spun at 1,000xG for 10 minutes at 4C using a swing bucket rotor. The supernatant was removed and the nuclei were resuspended in 45 uL of PBS with 0.5% BSA and RNAase inhibitor by pipetting. 2uL of the resuspended nuclei were diluted and counted using a hemocytometer based on Hoechst fluorescence. 18uL at a concentration of 1,000 nuclei/uL were provided to the Janelia Research Campus Quantitative Genomics core and run through the 10X Genomics Chromium Single Cell 5' (v2 Chemistry Dual Index) pipeline. 18uL at a concentration of 1,000 nuclei/uL were provided to the Janelia Research Campus Quantitative Genomics core and run through the 10X Genomics Chromium Single Cell 5' (v2 Chemistry Dual Index) pipeline.