title: GSM7359848: Pooled PTGs from Gcm2-/- mice complemented with Pth-tdTomato mouse ESCs, biological replicate 2; Mus musculus; RNA-Seq
library_construction_protocol: We performed a series of experiments twice (each experiment was titled para10_215 and para10_222). Adult Gcm2-/- mice complemented with mESCs showing 50‒70% chimerism of peripheral blood cells were used (three for para10_215 and five for para10_222). A pair of mESC-derived PTGs was removed and placed in a 20-μl PBS drop. Isolated mESC-derived PTGs were cut into small pieces using Vannas scissors. Minced mESC-derived PTGs were transferred into a 1.5 ml siliconized tube (Watson). They were incubated in 25μl of papain solution (Worthington) for 15 min at 37°C under 5% CO2 in air. Twenty-five μl of 10 mg/ml collagenase XI (Sigma) / HBSS (Wako) supplemented with 120 μl of 170mM CaCl2 was added into the same tube, which was incubated for 15 min at 37°C under 5% CO2 in air. Finally, 10μl of 0.25% trypsin-EDTA was added. After 1 min, pipetting was done several times, yielding single cells that were suspended in 140 μl of medium containing equal volumes of 10% FBS/DMEM and ovomucoid protease inhibitor (Worthington) and placed on ice. Doublets were discriminated using FACSAria Fusion Flow Cytometer. Sorted single cells were collected in 500μl 0.1% BSA /PBS in a 1.5 ml siliconized tube. The cell suspension was centrifuged at 300g at room temperature for 5 min. Single-cell gel beads-in-emulsion were generated using the Chromium Controller (10x Genomics). Libraries were generated using the Single Cell 3' Reagent Kit v.3.1 for dual index (10x Genomics) according to manufacturer's instructions.