GSM7775149: Intestinal lamina propria lymphocytes, IL-22Fc, replicate 2; Mus musculus; RNA-Seq
mesenteric fat tissue and Peyer's patches were carefully removed and small intestine was opened longitudinally and sliced into small fragments roughly 2 mm in length. The tissue was incubated in 5 mM EDTA–PBS on ice for 90 min. Every 30 min, the tissue was shaken vigorously and the supernatant was collected as fraction 1 in a new conical tube. The final fraction (enriched for crypts) was washed twice in PBS, centrifuged at 300g for 5 min, and dissociated with TrypLE express (Invitrogen) for 5 min at 37 °C. The single-cell suspension was then passed through a 40-μm filter and removed dead cell by using Dead Cell Removal Kit (Miltenyi Biotec, 130-090-101) for scRNA-seq. After IEC was removed, the remaining tissue was then minced with scissors and dissociated in RPMI containing 2% FBS, Liberase (31.25μg/ml) and DNase I (50 μg/ml; Sigma) at constant shaking at 37 °C for 35 min (175 rpm). The digested tissue was then filtered through a 70-μm strainer to remove large debris. Viable lamina propria lymphocytes were collected at the interface of a 40%/80% Percoll/RPMI gradient (GE Healthcare). Removed dead cell by using Dead Cell Removal Kit for scRNA-seq. Library was performed according to the manufacter's instructions (10x 3' Chomium Connect)