GSM9497652: Clonally traced otic and epibranchial ganglia region and surrounding tissue, embryonic day (E) 11.5; Mus musculus; RNA-Seq
Embryos were harvested at E11.5 following maternal euthanasia by CO₂ overdose and cervical dislocation and were subsequently euthanized by decapitation. Mouse embryos were dissected from the uterus, sacrificed, and transferred to ice-cold Hank's Balanced Salt Solution (HBSS). Fluorescent reporter expression (tdTomato) was assessed using a fluorescence stereomicroscope. Embryos were dissected into different regions containing olfactory and forebrain; eye, trigeminal ganglia  and midbrain and hindbrain; otic and epibranchial ganglia region; and the entire cranial region. Dissected tissues were placed in dissociation buffer containing Collagenase P (2 mg/mL) (Roche, Sigma-Aldrich), TrypLE (1×), and DNase I (1 µL/mL) prepared in HBSS with calcium and magnesium. Samples were then incubated for 20 minutes at 37 C in a preheated shaking incubator set at 150 RPM. After tissue were dissociated to stop the enzymatic were aded ice-cold HBSS supplemented with 2% FBS. Cells were pelleted by centrifugation at 500 × g for 15 minutes at 4 °C, resuspended in HBSS containing 2% FBS, and kept on ice. Prior to downstream applications, single-cell suspensions were filtered through a 40 µm cell strainer.  Flow cytometry sorting was performed using FACSAria Fusion or FACSAria III instruments (BD) equipped with a 100 µm nozzle and a 1.5 neutral density filter, and controlled via FACSDiva software (BD). Cells (~5,000–10,000 per reaction) were processed using the 10X Genomics Chromium V3.1 kit. cDNA was synthesized and amplified by 12 PCR cycles. Gene expression libraries were prepared and indexed using Single Index Plate T Set A. CloneID sequences were enriched via nested PCR with Q5 polymerase, purified between steps using 0.8× AMPure beads, and all libraries were quantified with BioAnalyzer and/or Qubit.