Single-cell analysis of early chick hypothalamic development reveals that hypothalamic cells are induced from prethalamic-like progenitors.

Purpose: scRNA-Seq was performed to profile chicken hypothalamus development. Methanol-fixed chicken embryos of dissected hypothalamus at HH8, HH8, HH13, HH15/16, HH18/19, HH20/21 were re-hydrated as previously described. Briefly, cells were washed twice (3000 x g for 5 min at 4’c) and resuspended in RNAse-free PBS with 1% BSA and 0.5 U/ul RNAse inhibitor. Cells were then used for the 10x Genomics Chromium Single Cell System (10x Genomcis, CA, USA) using V3.0 chemistry per manufacturer’s instruction loading between 8,000 and 12,000 cells per run. Libraries were sequenced on Illumina NextSeq 500 with ~ 200 million reads per library. Sequenced files were processed through the CellRanger pipeline (v.3.10, 10x Genomics) using Ensembl Gallus gallus genome (GRCg6a, release 95) compiled with CellRange mkref function.