GSM8231954: ent-2KO mutant feed with L4440 HT115 control bacteria; Caenorhabditis elegans; RNA-Seq
Worms were washed three times with PBS and collected in a 1.5 mL tube. We then added 100 μl of homogenization buffer  and grind the worms with a pestle motor for 30 seconds on ice. To minimize nuclei adhesion on the surface, all the pestles, tubes and filters were pre-coated with a homogenization buffer or 1x PBS. 900 μl of homogenization buffer was added to wash the pestle, and the total 1mL homogenized sample was transferred into a 1mL Dounce tissue grinder (Wheaton 357538). The grinder was autoclaved overnight in a 220℃ oven to deactivate ribonuclease. After placing the grinder on ice, 20 strokes were applied using a loose pestle, followed by another 20 strokes using a tight pestle, while avoiding generating foams. The 1000μl samples were filtered through a cell strainer (35 μm) and then through a Flowmi cell strainer (BelArt, H13680-0040) into a new 1.5 mL tube. The tubes were centrifuged for 10 minutes at 1000 g at 4°C, and the supernatant was removed while not disturbing the pellet, which was hardly visible. The pellet was resuspended with 500 μl 1x PBS with 0.5% BSA and RNAase inhibitor and filtered with a Flowmi cell strainer (BelArt, H13680-0040) again into a 5 mL flow cytometer tube. A total of 20 μl was transferred into another flow cytometer tube and diluted with 180 μl 1x PBS with 0.5% BSA and RNAase inhibitor as an unstained control. Hoechst (Invitrogen 33342) was used to stain the nuclei in 1:1000 working concentration. We coated the 1.5 mL sorting collection tube with 1x PBS with 0.5% BSA and RNAase inhibitor and ~300,000 nuclei were sorted with Hoechst 33342 positive gating, which indicates DNA content, and forward scatter area (FSC-A) gating, representing the particle size above threshold. The intestinal nuclei, which are polypoid (32N), usually form an obvious cluster separating from the other 2N somatic nuclei, and these were also included in our collection. The collection tube was centrifuged at 800g for 8 minutes at 4℃ and the sheath buffer supernatant was carefully removed before resuspending the sorted nuclei with 40-50 μl 1x PBS with 0.5% BSA and RNAase inhibitor. The concentration and morphology of the nuclei were checked under a microscope to ensure high-quality nuclei isolation. If the results were desirable, we proceeded to generate gel emulsion with 10X Chromium Controller.For sorting the nuclei, we used Sony MA800 sorter. To obtain the best nuclei morphology and the least RNA degradation, it is recommended to minimize the sorting time. Two researchers could simultaneously set up the sorter and perform nuclei processing. Library preparation was carried out using the published 10X Chromium Single Cell 3' v2/v3 Solution protocol (Single/dual index). The resulting libraries were sequenced on the Novaseq 6000 platform, with the recommended cycle numbers: 26 cycles for Read 1, 8 cycles for i7 index, and 98 cycles for Read 2.