GSM8488966: Plucked Feather, 12 days, scRNAseq; Gallus gallus; RNA-Seq
Dissected samples were placed in PBS on ice and kept for less than 15 minutes before proceeding to single cell or single nuclear isolation. For samples from stage 38 embryos, we gently chopped the tissue with a scalpel before incubating the samples in a solution of Pronase (1mg/1ml PBS) for 60 minutes at 37˚ C. The tissue was then gently passed 20 times though an 18 gauge needle. The resulting cellular suspension was filtered through a 70μm filter and then filtered twice through a 40μm filter to remove larger debris. We then spun down the cell suspension at 300 g for 5 minutes removing the supernatant and resuspended the pellet in calcium and magnesium free PBS containing 1% bovine serum albumin twice to wash the cells. We used Trypan blue to estimate cellular viability with only a ≥ 70% viability used for further processing. We also estimated the cellular concentration on a hemocytometer before diluting the suspension to 1000 cells/μL to submit for 10x library preparation. For samples from stage 39 and 40 embryos, we resorted to nuclear isolation as single cell dissociation was impossible due to the keratinization of cells in these stages. Our protocol was adapted from Sarropoulos et al. 2021.2 Plucked feathers were placed in 2 mL of TST lysis buffer containing 1ml 2x Salt-Tris Buffer (see below for 1x buffer), 60 μL 1% Tween-20, 10 μL 2% bovine serum albumin, and 930 μL nuclease free H2O. We ground the tissue using a dounce homogenizer grinding 20 times with the loose pestle and 20 times with the tight pestle. This suspension was filtered through a 70μm filter and then filtered twice through a 40μm filter. Nuclei were pelleted at 500 g for 5 minutes before being washed with a Salt-Tris Buffer (146 mM NaCL, 1mM CaCL2, 21mM MgCl2, 20mM tris-HCl PH 7.5). The nuclei were then and resuspended in ice cold PBS without calcium or magnesium and containing 1% bovine serum albumin for submission for 10x library preparation. Library construction was performed according to the manufacturers instructions (single cell 3' v3 protocol, 10x Genomics).