GSM6616316: denervated_ileum_day0 IR #2; Mus musculus; RNA-Seq
Mouse ileum (5cm from the cecum) was removed; fat was trimmed off. Ileum was cut into 1-cm pieces, added to 10 ml Dissociation Reagent 1 [HBSS Ca2+ and Mg2+ free; 10mM HEPES; 10mM EDTA; 1.5mM DTT; 5% FBS] and incubated on ice for 20min. Samples were transferred to gentleMacs C-tube containing 10 ml Dissociation Reagent 2 [HBSS w/o; 10mM HEPES; 10mM EDTA; 5% FBS] and incubated on ice for 1hr, followed by 1min spin at 100rpm on gentleMacs dissociateor (Miltenyi Biotec). Samples were washed 2x in RPMI, followed by pelleting at 100g in a swing bucket at 4 °C. After the last spin, samples were resuspended in RPMI. To better preserve epithelial cells, samples were separated by free-floating villi chucks and undissociated piece (containing crypts and mesenchymal tissue). The undissociated pieces were picked out and placed in a new C-tube containing 2.5mL digestion buffer w/ collagenase [HBSS, 10mM HEPES, 5% FBS, 0.8 mg/ml dispase, 0.2 mg/ml Collagenase P; 0.1 mg/ml DNAse]. The villi chucks were spun again at 100g, and resuspended in 2.5mL digestion buffer w/o collagenase [HBSS, 10mM HEPES, 5% FBS, 0.8 mg/ml dispase, 0.1 mg/ml DNAse]. Both tissue segments were incubated in 37°C water bath for 5min, followed by 1 min spin at 100rpm on gentleMacs dissociateor. Two portions of samples were combined again, washed by RPMI, passed through 100um filter, spun at 300g to pellet single cell and resuspended in PBS + 3% FBS. Cells were filtered with 40um filter and counted right before 10x chromium chip loading. Sample processing for single-cell RNA-seq was done using Chromium Single Cell 3' Library reagent kit v3.1 following manufacturer's guide (10x Genomics). Cell density and viability of single-cell suspension were determined by Vi-CELL XR cell counter (Beckman Coulter) or by manual counting using a hemocytometer. We loaded ~10,000 cells on each chip. cDNAs and libraries were prepared following the manufacturer's user guide (10x Genomics). Libraries were profiled by Tapestation D1000 kit (Agilent) and quantified using IQubit HS dsDNA kit (Invitrogen). Each library was sequenced in one lane of HiSeq4000 (Illumina) following the manufacturer's sequencing specification (10x Genomics). Chromium Single Cell 3' Library reagent kit v3.1