GSM7877349: Control, E11.5, Sorted CNCCs; Mus musculus; RNA-Seq
Single-cell dissociation, cell sorting, and sequencing: To isolate CNCCs, E11.5 embryonic heads were cut just below pharyngeal arch 2 and the otic vesicle by microdissection. Using Wnt1:CRE-mediated recombination of the mTmG reporter allele (Muzumdar et al., 2007), CRE-positive embryos were selected for further processing. Tissue samples were then subject to a cold protease single-cell dissociation protocol (Adam et al., 2017), with modifications. Embryo heads were incubated in a protease cocktail comprised of Bacillus licheniformis Subtilisin A (Creative Enzymes), Accumax, and Accutase (Fisher Scientific) suspended in DEPC-PBS as previously described (Sekiguchi and Hauser, 2019). The tissue was then carefully disrupted using a disposable Eppendorf pellet pestle and placed on a rotator at 4°C for 30 min. During this step, samples were gently triturated every 10 min using a wide bore 1 mL pipette tip. The enzymatic reaction was quenched using an equal volume of 10% heat-inactivated FBS (Fisher Scientific) in 1X phenol red-free DMEM (ThermoFisher). Dissociated cells were washed thrice in DEPC-PBS by centrifugation at 600 x g at 4°C for 5 min and then transferred, through Bel Art SP Scienceware Flowmi 40-μm strainers (ThermoFisher), into chilled flow tubes precoated with DEPC-PBS. After GFP-positive sorting on the University of Iowa Flow Cytometry Facility's ARIA II system (Becton Dickinson), CNCCs were re-suspended in DEPC-PBS containing 0.04% non-acetylated BSA (New England Biolabs). Cell viability was determined to be >95% by Trypan Blue staining. During cell sorting, genotypes were confirmed by rapid genotyping, allowing isolation of GFP-positive cells from two Tfap2HET control and two sibling-matched Tfap2NCKO mutant embryos. Cells from each “biological replicate” were combined to generate a single control and mutant sample. ~6,000 GFP-positive CNCCs from each condition were subject to library preparation by the University of Iowa Genomics Division on the 3' expression scRNA-seq 10X Chromium v3.1 pipeline. Libraries were sequenced on an Illumina NovaSeq 6000 platform as 100-bp paired-end reads. Over 20,000 reads per cell were acquired, resulting in ~1.5 x 108 reads per condition.