NextSeq 500 sequencing; Single cell RNA-seq of E10.5 mouse barrelette progenitors
To collect rhombomere 3 (r3)-derived progenitors from E10.5 K20tdTomato/+ mouse, r2 – r4 regions were micro dissected. Dissected tissue was kept in PBS 1X containing 2μM  actinomycin D (Sigma, A1410) for 10 minutes on ice, then treated with papain digestion mix (papain 10mg/ml/ cysteine 2.5mM/ HEPES pH7.4 10mM/ EDTA 0.5mM/ DMEM 0.9X/ 40μM actinomycin D) for 3 minutes at 37°C and immediately put on ice.  Tissue was rinsed by ice-cold DMEM 1X containing 2uM actinomycin D, and dissociated by pipetting and filtered. r3-derived cells were collected by FACS. Not Applicable. 50,000 cells were sorted directly into 50µl of PBS-0.04%BSA. Cell concentration was determined using a TC20 automated cell counter (BioRad). 4,500 and 2,250 cells were respectively loaded into 2 different channels of a Chromium Single Cell A Chip (10X genomics). Reverse-transcription, cDNA pre-amplification (13 cycles) and library preparation were performed according to the manufacturer instructions (Chromium Single Cell 3′ Library & Gel Bead Kit v2, 10X genomics). 