Single cell RNA sequencing (scRNAseq) on mouse embryonic cranial motor neurons and surrounding tissue from wildtype and HCFP1 cRE1dup/+ mice.

We found hereditary congenital facial paralysis type 1 (HCFP1) is caused by variants that duplicate or change cis regulatory elements (cREs) controlling the neuronal expression of the GATA2 transcription factor. We generated an HCFP1 mouse model in which tandem copies of the human version of a GATA2 regulatory element we term cRE1 were inserted proximal to the mouse cRE1 (cRE1dup/+; hg19: chr3:128,175,708-128,176,563). We report the use of single cell RNA sequencing (scRNAseq) for transcriptomic analysis of developing rhombomere 4(r4)-derived wild type (WT) and cRE1dup/+ facial branchial motor neurons (FBMNs) and inner ear efferent neurons (IEEs), as well as surrounding tissue, from embryos aged 9.5-12.5 post coitus (E9.5-12.5). scRNAseq (10x Genomics) libraries were generated from FAC-sorted WT and cRE1dup mouse embryonic cranial motor neurons spanning r3 (trigeminal motor neurons) to r7 (hypoglossal and glossopharyngeal motor neurons) marked by the motor neuron-specific Isl1MN-GFP transgenic reporter. GFP negative cells from the surrounding tissue were included for transcriptional and anatomic context. Cells were collected at E9.5 (n= 17 WT, 18 cRE1dup/+ embryos from 2 litters), E10.5 (n=12 WT, 7 cRE1dup/+ embryos from 2 litters), E11.5 (n=5 WT, 2 cRE1dup/+ embryos from 1 litter), and E12.5 (n=3 WT, 5 cRE1dup/+ embryos from 1 litter). Single cell cDNA libraries were generated using a Chromium Next GEM Single Cell 3? Library Kit v3.1. Libraries from littermate WT and cRE1dup/+ preparations were sequenced on common flow cells on a NextSeq 500 DNA sequencer using a NextSeq 500/550 High Output v2.5 kit (75 cycles) (Illumina). Raw sequence data was processed, filtered and aligned on the Cell Ranger analytic pipeline (10x Genomics) to generate FASTQ and filtered feature bc matrix files for deposit.