GSM9365226: d75_thymus_2; Sus scrofa; RNA-Seq
Fetal liver tissues were weighed and dispersed into single cell suspensions using glass homogenizers. Cells were then filtered using 100 µm cell strainers, washed with PBS, and treated with an ammonium chloride-based lysis buffer to remove red blood cells. After counting, 6 million cells from each liver were Fc-blocked with rat IgG (Sigma Aldrich, St. Louis, MO) and stained with anti-porcine CD45 antibody (ThermoFisher Scientific, Waltham MA). Cells were then stained with the propidium iodine (PI) viability dye and sorted for live CD45+ and CD45- cells using a BD FACS Melody cell sorter (BD Biosciences, Franklin Lakes, NJ). Thymus tissues were incubated in 3 ml of Hanks' Balanced Salt Solution (HBSS) with 2ul of collagenase Type 1 (Worthington, Lakewood, NJ) at 37°C for 30 minutes after which they were dispersed into single cells using glass homogenizers, washed with PBS, filtered, counted, stained with PI, and finally sorted for live cells. Bone marrow was collected from the scapula, femur, tibia, humerus, ulna, and radius of 47 DG fetuses, from the femur and tibia of 60, 75, and 90 DG fetuses, and from the ribs and sternum of 1 DOA and 7 MOA pigs after they were cut open with scissors. Cells were filtered, washed with DMEM containing 10% FBS, counted, stained with PI, and FACS sorted for live cells. Gene expression libraries were prepared using the Chromium Next GEM Single Cell 5′ Kit v2 (10× Genomics, Pleasanton, CA) according to the manufacturer's instructions. V(D)J libraries were enriched for TCR and BCR transcripts with pig-specific primers.