Single-nucleus transcriptomic brain atlas of Ceratina calcarata. Ceratina calcarata

Two samples of six brains of adult female small carpenter bees (Ceratina calcarata) were prepared for single-nucleus transcriptomic analysis. Bees were field collected in Toronto, Canada and dissected brains were flash-frozen in liquid nitrogen. The first sample was collected in January 2022 when females were overwintering and in diapause (winter). The second sample was collected in June 2022 when females were foraging and reproductive (summer).To isolate nuclei and prepare libraries for sequencing, each sample was processed at the Princess Margaret Genomics Centre in Toronto, Canada. Frozen tissue of each sample was cut into 1-2mm cubed portions using a chilled razor on dry ice. The portions of tissue were then covered with lysis buffer (0.32mM sucrose, 5mM CaCl2, 3mM Mg(Ac)2, 20mM Tris-HCl 7.5, 0.1% Triton X-100, 0.1mM EDTA 8.0, 40U/mL RNase Inhibitor, and water) and cut into smaller pieces and homogenized with glass douncers for five minutes. A portion of the sample was stained with SYBR green and nuclei quality was manually evaluated with disposable hemocytometers. The sample was then centrifuged at 800g for 10 minutes and resuspended in 2mL of wash buffer (1x PBS, 1% BSA, and 0.2 U/uL RNase Inhibitor). Then, the sample was centrifuged, resuspended, and centrifuged once more. Then, the sample was filtered through 40uM Flowmi cell strainer and transferred to a 1.5mL LoBind tube placed on ice. Nuclei were then counted.Samples were then loaded into 10X Genomics Gel-beads-in-emulsion wells on Chromium Chips following their protocol for Single Cell 3-prime Reagent Kits (v3). After dissolving the gel beads, nuclei were lysed, and mRNA was reverse transcribed to cDNA. Using PCR, the cDNA was then amplified to create libraries. Libraries were checked for quality using the Agilent Bioanalyzer High Sensitivity chip. The libraries were then sequenced on one of four lanes on a flow cell with an Illumina Novaseq 6000 to create base call files.Base call files were input to Cell Ranger (version 6.1.2) to generate fastq files using the function mkfastq.