GSM9449269: TCp, GEX; Mus musculus; OTHER
mLN and liver from P7 RorcVenus-creERT2 mice (n = 9); mesenteric anlagen, fetal liver, and fetal bone marrow from E17.5-18.5 RorcVenus-creERT2 mice (n = 25); mLN, liver, and bone marrow from P7 Il7rcreR26lsl-tdTomato mice  (n = 4), and mesenteric anlagen, fetal liver, and fetal bone marrow from E17.5-18.5 Il7rcreR26lsl-tdTomato mice (n = 9) were processed for Flex scRNA-seq analysis of progenitors (Supplemental Table 1). Fetal liver, liver, fetal bone marrow and bone marrow were depleted of lineage (TCRb, TCRgd, CD19, B220, NK1.1, Ter-119, Ly-6G, CD3e)+ cells via staining with biotinylated antibodies followed by magnetic bead (Dynabeads M-280 streptavidin; Invitrogen) negative selection. Cells were incubated with anti-CD16/32 and extracellular antigens were stained for 30 minutes at room temperature in sorting buffer (2% FBS, 2mM EDTA, in PBS) and labeled with BioLegend TotalSeq-C Hashtag antibodies. Cells were washed and resuspended in cRPMI with SYTOX blue (Invitrogen) for exclusion of dead cells. Cells were sorted as per Extended Data Fig. 3a into 500ml of Fixation Buffer (4% Formaldehyde, 1X Conc. Fix and Perm Buffer – 10x Genomics PN2000517). Cells were fixed for 16-20h at 4°C. To stop the fixation, cells were spun down at 850 rcf for 5 min at room temperature and quenched with 500ml of Quenching Buffer (1X Conc. Quench Buffer – 10x Genomics PN-2000516). Preparations were then processed by adding 0.1 volumes of Enhancer (10x Genomics PN2000482) and 10% glycerol for storage at -80°C.  Samples were then thawed at room temperature, centrifuged at 850 rcf for 5 min and resuspended in 1 mL 0.5X PBS – 0.02% BSA. Cell concentration and viability were assessed by 0.2% (w/v) Trypan Blue staining (Countess II). Cells were processed per hybridization according to the 10X protocol. Hybridizations were set up in 80 mL of hybridization mix with 20 mL of Human or Mouse WTA probes (10x Genomics PN-2000510 or PN-2000718) and 4 mL of Antibody Multiplexing Barcode (10x Genomics PN-2000917- 2000932). Hybridizations were performed at 42°C for 16-24h. After hybridization samples were diluted in Post-Hyb Wash Buffer and measured by 0.2% (w/v) Trypan Blue staining (Countess II). For each experiment, we pooled an equal number of cells from each hybridization to have an equal contribution per sample. Pooled cells were then washed 3 times in Post-Hyb Wash Buffer for 10 min at 42°C, resuspended in Post-Hyb Resuspension Buffer, filtered through a Miltenyi Biotec 30 mm filter, and measured with the cell counter to determine the amount required for the Chromium X run. GEM encapsulation was performed according to the 10X Genomics protocol. After loading the Chip Q and running it on the Chromium X, GEMs were recovered and processed as indicated by 10X Genomics. Post-processing, the product was pre-amplified and indexed to construct the sequencing library. All libraries were sequenced on an Illumina Novaseq X with standard dual indexing and demultiplexing.