GSM8455325: Md, fmi, 13.5dpc, Md18II1; Monodelphis domestica; RNA-Seq
All tissue was processed as follows, with small species-specific modifications as noted. Tissue was minced with a scalpel into ~1 mm3 cubes in 2 mL of digestive solution containing 0.2 mg/mL Liberase TL (05401020001, Sigma). Tissue suspensions were heated at 37°C for 15 minutes and then passed 10 times through a 16-gauge needle attached to a 3-mL syringe. This incubation and needle passage process was repeated two more times, with the substitution of an 18-gauge needle. Finally, 2 mL of charcoal-stripped fetal bovine serum (100-199, Gemini) was added and the suspension was immediately passed through a 70-μm cell strainer then a 40-μm cell strainer. The flowthrough was pelleted by centrifugation at 500 g for 5 minutes and cells were resuspended in 1x ACK red blood cell lysis buffer (A1049201, Thermo-Fisher), incubated at room temperature for 5 minutes, centrifuged again and resuspended in PBS containing 0.04% bovine serum albumen (A9647, Sigma). At this point, cells were examined on a hemocytometer to gauge concentration and check for debris and cell death using trypan blue stain (15250061, ThermoFisher). Mouse tissue was processed as above with the addition of Accumax (Stem Cell Technologies) to the final 0.04% bovine serum albumen solution. Guinea pig tissue was processed as with the mouse with the following modifications: a wide-bore 1 mL pipette tip was substituted for the 16-gauge needle, and 0.2 mg/mL collagenase I (17018029, Thermo-Fisher) was added to the digestion solution. As tenrec and guinea pig tissue showed higher cell death, a fractionated protocol was adopted to reduce exposure of cells to digestive enzymes. For these species, cells which had dislodged following each passage step were separated from intact tissue and immediately passed through a 70-μm cell strainer into 2 mL of charcoal-stripped fetal bovine serum (100-199, Gemini), while remaining intact tissue was allowed to continue with further digestion. Each fraction was centrifuged at 300 rpm for 5 minutes and resuspended in 500 μL ACK red blood cell lysis buffer (A1049201, Thermo-Fisher) for 5 minutes, then centrifuged at 500 rpm for 2 minutes and resuspended in 0.04% bovine serum albumen (A9647, Sigma) and kept on ice until dissociation was complete. Cells were counted on a hemocytometer and recombined in a proportional manner before sequencing. Cells were captured using the 10X Chromium platform (3' chemistry, version 3) Libraries were generated according to manufacturer protocols (CG000315: 10x Genomics 3' chemistry, version 3).