GSM3444964: Intact- scRNAseq samples 1 to 2 and Contralateral- scRNAseq samples 1 to 3; Ambystoma mexicanum; RNA-Seq
Animals were narcotized in 0.1% Tricaine. Intact limbs were amputated and placed into 420ul of 0.7X PBS with 5mM EDTA, 25mM cysteine, and 40ul papain (Sigma P3125) within a 1.5mL eppendorf tube. Tissue was minced with scissors in this solution for 3-5 min until tissue was completely broken down. 0.6mL of ice cold 0.7XHBSS was added to the cells and cells were dumped (not pipetted) onto a 70um filter held above a 6 well plate on ice. The filter was washed with 2mL ice cold 0.7X HBSS.  This plate was allowed to sit for 10 minutes on ice. Following, a 3mL syringe was used to slowly layer 3mL of 5% Optiprep (diluted in 0.7X HBSS) down the side of the well. Cells were then let to sit for 2 minutes, before carefully removing 3mL of the 0.7X HBSS with a p1000 pipette. These steps were then repeated with 10% and 15% Optiprep, removing the more dilute solution (top layer) after each step. At the end, around 200uL of 15% Optiprep should be left with the cells. A trypan blue count was taken to ensure viability greater than 90%, and 0.5% BSA by volume was added to the remaining cells. Animals were narcotized in 0.1% Tricaine. Following this, iridectomy scissors were used to remove the blastema at its interface with the stump. The removed blastema was placed in 400uL papain (Worthington LK003178, resuspended in 2.5mL 0.7X PBS) in an Eppendorf tube. The tube was placed on a nutating rocker for 30 minutes, with gentle pipetting performed every 5 minutes after an initial 10 minute incubation. After 30 minutes, the cells were carefully passed through a 70um cell strainer to a well of a 6 well plate on ice and 0.7X HBSS ice cold was used to collect any cells that remained in the tube and subsequently this was passed through the cell strainer into the 6-well plate. With 2mL 0.7X HBSS ice cold wash the cell strainer, making sure that 0.7X HBSS passing through the strainer enters the 6-well plate. This plate was allowed to sit for 10 minutes on ice to allow cells to settle. Following, a 3mL syringe was used to slowly layer 3mL of 5% Optiprep (diluted in 0.7X HBSS) down the side of the well. Cells were then let to sit for 2 minutes, before carefully removing 3mL of the 0.7X HBSS with a p1000 pipette. These steps were then repeated with 10% and 15% Optiprep, removing the more dilute sample after each step. At the end, around 200uL of 15% Optiprep should be left with the cells. A trypan blue count was taken to ensure viability greater than 90%, and 0.5% BSA by volume was added to the remaining cells. A detailed protocol can be found on protocols.io (dx.doi.org/10.17504/protocols.io.qmmdu46) TruSeq HT
GEO
GSM3444964