GSM6069861: Spider late germ-disc 2, snRNA-seq; Parasteatoda tepidariorum; RNA-Seq
The spider embryos were dechorionated with 100% commercial bleach, rinsed with distilled water, and transferred into CGBS-CMF (55 mM NaCl, 40 mM KCl, 10 mM Tricine, pH6.95)-0.01% (for cell) or 0.5% (for nucleus) BSA solution, where vitelline membrane was manually removed with forceps. For preparation of dissociated cells, the devitellinized sample was filtered through 40-mm cell strainer (pluriStrainer-Mini) and collected in a 1.5-mL DNA LoBind Tube (Eppendorf). For preparation of nuclei, the devitellinized sample was centrifuged in a 1.5-mL tube (Eppendorf) and suspended in 400 mL homogenization buffer (250 mM Sucrose, 10 mM Tris (pH 8.0), 25 mM KCl, 5 mM MgCl2, 0.1% Triton-X100, 0.1 mM DTT, 100 u/mL RNase Inhibitor (SUPERaseIn RNase Inhibitor: Invitrogen, AM2694), Protease Inhibitor at 1:100 dilution (Nacalai tesque, 25955-11)), where the sample was homogenized with a loose plastic pestle (fisher scientific) by 40 strokes. The sample was then centrifuged and suspended with CGBS-CMF-0.5% BSA-100 u/mL RNase Inhibitor solution, followed by filteration through the 40-mm cell strainer. The centrifugation and resuspension steps were repeated twice. Adding 4 volumes of CellCover (Anacyte Laboratories), the cell and nucleus samples were stored at 4 ˚C until use. The stored cells or nuclei were centrifuged and resuspended with the CGBS-CMF-based solution, and they were individualized using BD Rhapsody microwell-based platform, followed by library construction according to the manufacture's instructions. Construction of the single-nucleus RNA-seq library was performed with some modifications; RNase Inhibitor was added to the Sample buffer, and Proteinase K was added to the Lysis buffer, and the lysis step was prolonged from 2 minutes to 5 minutes. Single-cell and single-nucleus RNA-seq