title: GSM5368455: Ileum_adult (key metabolic tissues dataset); Bos taurus; RNA-Seq
library_construction_protocol: The rumen, reticulum, omasum, abomasum, ileum, and rectum tissues isolated from lactating cows were stripped of the outer muscle layers, and then minced into 10×0.5mm2 pieces on ice with scissors. The tissues were incubated in a 37℃ water bath with 20 mM EDTA for 30 min, and then were rinsed with DPBS and chopped into 1 mm pieces. Tissue pieces were transferred to a 15 mL centrifuge tube, and re-suspended with 0.25% Trypsin-EDTA (Gibco). After incubated in a 37℃ water bath for 5 min, the centrifuge tube containing tissues was inserted into ice for 2min, and pre-chilled HBSS was added to stop the digestion. After centrifuged at 300 × g for 2 min at 4℃, the supernatant was discarded, and samples were washed twice with cold HBSS and then re-suspended with dissociation enzymes. Samples were treated with different enzymes for different durations (for rumen, reticulum, omasum, and abomasum tissues: 1.5mg/ml collagenase I, 1.5ml/ml collagenase IV,1.5mg/ml dispase, 100U/ml hyaluronidase, and 50U/ml DNase I for 30 min at 37℃; for ileum tissue:4.5ml Buffer X, 50μl enzyme P, 50μl buffer Y, 100μl enzyme D, and 20μl enzyme A of Multi Tissue Dissociation Kit 2 (Miltenyi biotec, Bergisch Gladbach, Germany); for rectum tissue: 1mg/ml collagenase/dispase for 41 min at  37℃). For salivary gland samples, the isolated tissues from parotid gland were transferred into cold 1×PBS, blood and fat were removed, and chopped into 0.5 mm pieces and then incubated with 1.5mg/ml collagenase II, 1.5mg/ml dispase, and 50U/ml DNase I for 20 min at 37℃. For liver samples, the isolated tissues were transferred into cold DPBS, minced into 1mm pieces, and then incubated with 1.5mg/ml collagenase I, 20U/ml papain, and 50U/ml DNase for 20 min at 37℃.For the mammary gland sample, the isolated tissues were were cut into 5 mm pieces and washed using 1 × PBS (with 5% FBS) after removing the adipose tissue. Subsequently, samples were bath with 20 mM EDTA (with 5% FBS) at 100 rpm for 20 min at 37°C, and then incubated with dissociation enzymes (Dataset S1). The mixture was passed through 70 μm and 30 μm SmartStrainers and transferred into a new EP tube, and centrifuged at 300 × g for 5 min at 25°C. After removing the supernatant, the cells were washed with 3 mL HBSS with 5% FBS and centrifuged at 300 g for 5 min at 25°C.For the peripheral blood sample, PBMC and neutrophil were enriched by Ficoll (HY2015, TBD) separation. The single-cell RNA-seq library preparation was performed using the Chromium Single Cell 3' Reagent Kits v3.