GSM5765755: Duodenum newborn; Bos taurus; RNA-Seq
After removed the outer muscle layers, the adult (AD) duodenum, jejunum, cecum, and colon samples were chopped into 1 mm pieces and treated with different enzymes for different durations：the duodenum and cecum samples were dissociated using the Lamina Propria Dissociation Kit (Miltenyi biotec, Bergisch Gladbach, Germany) for 30 min at 37℃; the jejunum and colon samples were dissociated using the Multi Tissue Dissociation Kit 2 (Miltenyi biotec, Bergisch Gladbach, Germany) for 41 min at 37℃. Then, the digestion was stopped by adding 10% of FBS, followed by a filtration step with 40 μm sieve. Next, after centrifuged at 300 × g for 5 min at 4°C and washed with 1× DPBS containing 2% FBS and 0.2 mM EDTA, the samples were treated with Red Blood Cell Lysis Solution (Miltenyi Biotech, Bergisch Gladbach, Germany) for 2 min. The samples were washed with 1× DPBS containing 2% FBS and 0.2 mM EDTA, centrifuged at 300 × g for 5 min at 4°C, and re-suspended in 1× DPBS containing 10% FBS and 0.2 mM EDTA.  For the NB reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, colon, and rectum samples, we firstly removed the outer muscle layers of samples, and then minced samples into 10 × 0.5 mm2 pieces. The samples were incubated with 20 mM EDTA for 30 min at 37℃, and then rinsed with DPBS and chopped into 1 mm pieces. Tissue pieces were incubated in a 37℃ water bath for 5 min with 0.25% Trypsin-EDTA (Gibco) and then the centrifuge tube containing tissue pieces was inserted into ice for 2 min, and the digestion was stopped by adding the pre-chilled HBSS. The supernatant was discarded after centrifuged at 300 × g for 2 min at 4℃, and samples were washed twice with cold HBSS. Samples were incubated with different dissociation enzymes for different durations. For NB reticulum, omasum, and abomasum samples, the multiple enzymes (1.5 mg/ml collagenase I, 1.5 mg/ml collagenase IV, 100 U/ml hyaluronidase, 1.5 mg/ml dispase, and 50 U/ml DNase I) were added and treated for 30 min at 37℃. For the NB duodenum, jejunum, ileum, cecum, colon, and rectum samples, the multiple enzymes in the Propria Dissociation Kit (Miltenyi biotec, Bergisch Gladbach, Germany) were added and treated for 30 min at 37℃. The 10% FBS was added to stop the digestion, followed by a filtration step through 70 μm and 30 μm SmartStrainer (Miltenyi biotec, Bergisch Gladbach, Germany). After centrifuged at 300 × g for 5 min at 4℃, samples were then re-suspended in 2 mL of HBSS. Then, samples were centrifuged at 300 × g for 5 min at 4℃ and washed twice with 1×PBS with 0.04% BSA. Finally, samples were centrifuged at 300 × g for 5 min at 4℃ and re-suspended in 1×PBS with 0.04% BSA.   For the NB liver sample, the isolated sample was transferred into cold DPBS, minced into 1mm pieces, and then incubated with 1.5mg/ml Collagenase I, 20U/ml papain, and 50U/ml DNase I for 20 min at 37℃. The dissociated cells were then passed through 70 μm and 30 μm SmartStrainer, centrifuged at 300 × g for 5 min at 4℃, and then re-suspended in 2 mL of cold HBSS. After centrifuged at 300 × g for 5 min, dissociated cells were washed twice with 1×PBS with 0.04% BSA at 4℃ and then re-suspended in 1×PBS with 0.04% BSA.  For the NB salivary gland sample, the isolated sample from parotid gland was transferred into cold 1×PBS. The blood and fat of the sample were removed, and then the sample was chopped into 0.5 mm pieces. After incubated with 1.5mg/ml collagenase II, 1.5mg/ml dispase, and 50U/ml DNase I for 20 min at 37℃, the sample was passed through 70 μm and 30 μm SmartStrainers, and then centrifuged at 300 × g for 5 min at 4℃. Dissociated cells were re-suspended in 300 μL of RPMI 1640 medium (Gibco), and 3mL of Red Blood Cell Lysis Solution wad added for 3 min. Dissociated cells were then centrifuged at 300 × g for 5 min at 4℃ and re-suspended in 2 mL of RPMI 1640 medium. After centrifuged at 300 × g for 5 min, dissociated cells were washed twice with 1×PBS with 0.04% BSA at 4℃ and then re-suspended in 1×PBS with 0.04% BSA. The single-cell RNA-seq library preparation was performed using the Chromium Single Cell 3' Reagent Kits v3.