GSM4792393: Newborn_MG; Bos taurus; RNA-Seq
Animals were euthanized by cervical dislocation using a sharp knife in previously numbed individuals within a surgery room to obtain a ~10 g MG sample. Samples then were rinsed in DBPS and quickly transferred into MACS Tissue Storage Solution (NO. 130-100-008, Miltenyi Biotec, Germany) which was stored in a 37°C vacuum container and was quickly taken to the lab. The dissociation of MG was done according to published studies with some modifications described here. In brief, the MG tissue samples were placed in a sterile RNA-free cell culture dish with 1 × PBS (with 5% FBS) and were cut into 5mm pieces. The tissues were then washed using 1 × PBS (with 5% FBS) after removing non-targeted tissues such as white adipose tissue. Subsequently, samples were gently placed in 20mM EDTA (with 5% FBS) which had been incubated at 37°C at 100rpm for 20min. After that, samples were digested with 25U/ml hyaluronidase, 0.5mg/mL CollagenaseⅡ, 0.5 mg/mL Collagenase IV, 25 U/ml DNAse I and 5% FBS in HBSS for 30 min at 37 °C. The mixture was strained through a 70-30 μm cell strainer and transferred into a new EP tube, centrifuged at around 25°C, 300 × g for 5 mins. After removing the supernatant, the cells were washed with 3 mL HBSS (with 5% FBS) and centrifuged at around 25°C at 300 g for 5 mins. The Countess II Automated Cell Counter (AMQAX1000, ThermoFisher, USA) was applied to measure cell number and viability. The requirement for cell viability is greater than 90%. If the viability for isolated cells was lower than the threshold, we used MACS Dead Cell Remove Kit (NO. 130-090-101, Miltenyi biotec, Germany) to remove dead cells by following the manufacturer's recommendations. Single-cell RNA-Seq libraries were constructed as per the 10x Genomics protocol using GemCode Single-Cell 3' Gel Bead and Library V3 Kit." Single-Cell RNA-Seq