GSM8779763: Germ-free C57BL/6 mice, control group; Mus musculus; RNA-Seq
All colon tissues, from proximal to distal, were cut open longitudinally and washed in ice-cold PBS twice. The colon tissues were then immersed in ice-cold RPMI-1640 media and processed for tissue dissociation. Cecum was isolated from the gastrointestinal tract by performing a horizontal dissection at the junction between the cecum and the ileum, and between the cecum and proximal region of colon. The weight of the cecum in each group was measured. Dissected colon tissues from PBS control (n=5) and Fn-treated (n=5) GF mice were pooled for each group. Colon tissues were cut into approximately 5 mm pieces and washed three times with ice-cold PBS. The tissue fragments were pre-digested twice for 20 min in Hanks' Balanced Salt Solution (HBSS, Gibco, USA) without Ca2+ and Mg2+ containing 5 mM EDTA, 5% fetal bovine serum (FBS), 1 mM DTT at 37°C using a rotator mixer (Miltenyi Biotec, USA). The fragments were further transferred into a C-tube (Cat# 130-096-334, Miltenyi Biotec, USA) containing HBSS with Ca2+ and Mg2+ (Gibco, USA) and an enzyme mix (enzyme D, enzyme R, enzyme A) prepared from the mouse Lamina Propria Dissociation Kit (Cat# 130-097-410, Miltenyi Biotec, USA). The mixture was churned by gentleMACS Octo Dissociator (Cat# 130-134-029, Miltenyi Biotec, USA) with heaters for 30 min at 37°C. After incubation, ice-cold PB buffer (PBS containing 0.5% bovine serum albumin) was added to the C-tubes and the samples were passed through the 100 μm strainer. The samples were centrifugated at 300 x g for 10 min at 4°C and the pellet was resuspended in ice-cold PB buffer. The samples were filtered through a 70 μm strainer, and rinsed once with ice-cold PB buffer. Viable cells were subsequently purified using the Dead Cell Removal Kit (Cat# 130-090-101, Miltenyi Biotec). Single-cell suspensions were processed using 10X Chromium Next GEM Single Cell 5' RNA library v2 and 10X Chromium Next GEM Single Cell VDJ library v2 kit.