title: GSM9565751: King nuclei, whole body; Zootermopsis nevadensis; RNA-Seq
library_construction_protocol: Dissociation and isolation of single nuclei from the whole body of each sample was performed using an adapted version of a protocol established for Drosophila 36. Briefly, 100 μl of homogenisation buffer was dispensed into an DNA LoBind 1.5 ml tube (Eppendorf 022431021) and placed on ice. The homogenisation buffer consisted of nuclease free water, 250 mM nuclease free sucrose (FUJIFILM Wako Pure Chemical Industries, Ltd. 19-0001), 10 mM Tris (pH 8.0) (Nippon Gene 312-90061), 25 mM KCl (Cayman Chemical Company 600213), 5 mM MgCl2 (Serva 39772), 0.1% Triton-x 100 (FUJIFILM Wako Pure Chemical Industries, Ltd. 168-11805), 0.5% RNasin Plus (Promega N261B), 1× protease inhibitor (Promega G6521), and 0.1 mM DTT (Formedium Ltd. DTT005). Samples were placed into the homogenisation buffer and nuclei were released by approximately 20 gentle strokes of a plastic pestle (Violamo 1-2955-01) and an additional 900 μl of homogenisation buffer was added to the tube. Each sample was centrifuged (Eppendorf 5424R) at 1000 g for 10 minutes at 4°C, the supernatant was discarded, and the nuclei were resuspended in a 1000 μl of resuspension buffer on ice. The resuspension buffer consisted of 1× PBS (pH 7.4) (Thermo Fisher 2669978), 0.5% BSA (Wako Pure Chemical Industries, Ltd. 017-21273), and 0.5% RNasin Plus (Promega N261B). Each sample was then passed twice through a 100 µm cell strainer (As One 100-98-5) and placed into a 5 ml FACS tube (Falcon 352235) with a 35 µm cap for FACS sorting. Following dissociation, nuclei were stained with 1 μl of DAPI and were isolated from remaining debris with Fluorescence Activated Cell Sorting (FACS) using a BD FACSAria III. The worker sample was sorted into a 1.5 ml tube containing 400 μl of resuspension buffer. The queen, soldier, and king samples were sorted into 1.5 ml tubes containing 200 μl of resuspension buffer. Immediately after FACS sorting, nuclei were centrifuged at 1000 g for 10 minutes at 4°C and the 700 μl of supernatant was discarded. Nuclei were resuspended in the remaining 50-60 μl of supernatant, and the concentration of nuclei was calculated using a Thoma cell counting plate (Watson 177312C) with a ZEISS Axio Scope A.1 microscope. Sequencing was performed as per the 10x Genomics User Guide (CG000315 Rev E). Gel Beads-in-emulsion (GEMs) were generated using the Chromium X Instrument (10x Genomics, Pleasanton, CA, USA), and scRNA-seq libraries were constructed using the Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1 (Dual Index). The worker sample was loaded in the Chromium Next GEM Chip G with a target output of 10,000 nuclei, and the queen, soldier, and king samples were loaded in the Chromium Next GEM Chip G with a target output of 5,000 nuclei. All reactions were performed in the Eppendorf Mastercycler X50s with a 96-Deep Well Reaction Module. For both cDNA amplification and sample index PCR, 14 cycles were used across all samples.Sequencing was performed in-house on the NovaSeq 6000 platform