GSM9093323: ILC2s, Hp infected mice, biol rep 3; Mus musculus; RNA-Seq
Small intestinal single cell suspensions were prepared from the proximal 1/3 of tissue.Luminal contents were flushed with PBS, Peyer's patches removed, and tissue cut open. Pieces were incubated with epithelial cell extraction buffer (EEB: Ca2+/Mg2+ free HBBS, 3% FCS, 10 mM HEPES, 10 mM EDTA, 10 mM DTT) at 37℃ with gentle shaking for 15 min. Tubes were vortexed at full speed for 30 sec and supernatants discarded. The tissue was placed with fresh EEB at 37℃ for 15 min with gentle shaking, then vortexed for 30 sec and supernatant discarded. Tissue was placed in rinse buffer (Ca2+/Mg2+ sufficient HBBS, 3% FCS, 10 mM HEPES) and incubated at 37℃ with gentle shaking for 5 min. Tissue was placed in GentleMACS C tubes (Miltenyi Biotec) with digestion buffer (DB: Ca2+/Mg2+ sufficient HBBS, 3% FCS, 10 mM HEPES, 30 ug/ml DNase I, 0.1 Wunsch/ml Liberase TM), minced with scissors, and incubated at 37℃ with gentle shaking for 10-15 min. Single-cell suspensions were obtained using a gentleMACS tissue dissociator (Miltenyi Biotec), running program m_intestine_0, and then pouring through a 100 um filter and washing with FACS buffer (PBS, 3% FCS) Library was performed according to the manufacter's instructions (Chromium GEM-X Single Cell 3' Kit v4, 10x genomics)