GSM8745695: Spider early germ-band, scRNA-seq; Parasteatoda tepidariorum; RNA-Seq
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) containing 0.01% BSA (for cells) or 0.5% BSA (for nuclei). The vitelline membrane was then manually removed with forceps. For cell dissociation, the devitellinized embryos were incubated in CGBS-CMF containing 1 mg/mL erastase for 5 minutes at room temperature. Subsequently, the cell suspension was filtered through 70-mm and 10-mm cell strainers (pluriStrainer-Mini) and collected in a 1.5-mL DNA LoBind Tube (Eppendorf). Finally, BSA was added to a final concentration of 1%. For nuclei isolation, the devitellinized embryos were centrifuged in a 1.5-mL LoBind tube (Eppendorf) and resuspended 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)). The sample was then homogenized with a loose plastic pestle (fisher scientific) by 40 strokes. The nucleus sample was subsequently centrifuged and resuspended with CGBS-CMF-0.5% BSA-100 u/mL RNase Inhibitor solution, followed by filtration through the 40-mm cell strainer. This centrifugation and resuspension steps were repeated twice. Finally, 4 volumes of CellCover (Anacyte Laboratories) were added to both the cell and nucleus samples, which were then stored at 4 ˚C until further 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.