GSM7017775: Ngn3, scRNA; Mus musculus; RNA-Seq
Intestinal tissue was obtained from Neurog3-Cre; lsl-tdTomato mice (1 adult male), or Neurod1-Cre; lsl-tdTomato (3 adult females), cut longitudinally, washed (cold phosphate-buffered saline or PBS), cut into small ~5 mm pieces, and incubated (gentle agitation, 20 minutes, 4C) in EDTA solution (20 mM EDTA-PBS, Ca/Mg-free) in LoBind Protein tubes (Eppendorf 0030122216). The specimen was shaken, the tissue allowed to settle, and the supernatants collected. The residual tissue was again incubated similarly with EDTA solution, and supernatants were combined, and centrifuged (300 g, 5 minutes, 4C) Pellets were washed (2x, PBS (Ca/Mg-free) supplemented with 5% Fetal Bovine Serum (FBS), 4C) and incubated (37C, 2 minutes) in protease solution (TrypLE express, Thermo Fisher 12604013) supplemented with DNase (100 U/ml, Worthington Biochemical LK003172). The suspension containing dissociated cells was centrifuged (300 g, 5 minutes), washed (2x, PBS (Ca/Mg-free) containing 5% FBS, 4C) The resulting pellet was resuspended in FACS buffer (5% FBS in DMEM/F12, HEPES, no phenol red) containing DNase (100 U/ml), TO-PRO-3 (Thermo Fisher T3605, 1:10,000) to label dead cells, and Calcein Violet (Thermo Fisher 65-0854-39, 1:10,000) to label living cells. Cells were filtered (1 x 70-um, 1 x 40-um) and tdTomato+, Calcein Violet+, TO-PRO-3- cells were collected by fluorescence activated cell sorting using a FACS Aria (BD Biosciences). Collected cells were loaded into the 10x Genomics Chromium Controller, and cDNA prepared and amplified according to manufacturer's protocol (10x Genomics, Chromium single cell 3' reagent kit v3, 12 cycles per amplification step).