GSM7567344: SpiB replicate2; Mus musculus; RNA-Seq
Thymi from  two 15wk old Spib+/– and two Spib–/– chimeras were surgically removed following perfusion, then trimmed of fat and connective tissues, and placed in PBS on ice. Single cell suspensions were prepared by mechanical dissociation of the thymi through a 40μm strainer using a syringe plunger. Cells were washed with 10 ml MACS buffer. 25 million cells per sample were first stained with 5 μl anti-mouse CD16/CD32 in 50 μl labeling buffer (PBS + 1% BSA) for 10 min at 4 °C, then an antibody mix containing both 5' hashing antibodies and flow cytometry antibodies in labeling buffer was added to complete to ~100 μl total volume, and cells were incubated for 30 min at 4 °C. Cells were then washed 3 times with 5 ml labeling buffer. 10 minutes before each sample was sorted, it was incubated with a viability dye for 5 min at room temperature, then washed and resuspended in PBS + 2% BSA. CD45.1+CD3– (CD4/CD8DP)–MHCIImid-high cells were sorted into PBS supplemented with 50% FBS and then washed and resuspended in PBS + 0.04% BSA, counted, and volume was adjusted to a concentration of 1,000,000 cells/ml. Each two non-replicate samples were combined evenly into one pool. The 10x Genomics platform loading and library preparation were done according to the manufacturer's instructions, as previously described (10x Genomics) and were sequenced with paired-end 100-bp reads on an Illumina NovaSeq6000. 5' single cell RNA-seq