GSM6215324
Cells from each E10 control and cKo caudal half were resuspended in 50 µL FACS-buffer (1× PBS with 1 % BSA). Sorting antibodies were prepared and added to the cell suspension, and incubated for 30 min at 4 °C. After washing, each oligo barcoded streptavidin with titrated dosage (0.125 μL for 1 million cells) were incubated with the stained cells for 20 min at 4 °C. After 3 washes, 10nmol/L Biotin was added to bind redundant streptavidin in the cell suspension, and incubated for about 5min at 4 °C. After 2 washes in 0.1 % BSA/PBS, staining with 7-AAD was performed for 5 min. Cells were sorted with gating strategy shown in Fig. 4B on BD FACS Aria II and harvested in 200 μL PCR tube. Cells from each tissue were pooled into one 1.5 mL centrifuge tube. Cells were collected at 400× g at 4 °C for 5 min, resuspend in 50 μL 0.04 % BSA/PBS to load into one lane of a 10× Chromium V3 chip. The cDNA preparation was performed following the instruction manual, and the hashtag library was prepared following the BioLegend TotalSeq-B guide. The final cDNA library and tag library were both sequenced on NovaSeq 6000. Cells were sequenced to an average depth of 0.1 M per cell for cDNA and 0.01 M per cell for hashtags.