Transcriptome Analysis of Cranial Neural Crest Cells

We used 1500 GFP+ cranial neural crest cells per replicate to prepare libraries. cDNA libraries were prepared using the Takara Bio SMART-Seq v4 Ultra Low Input cDNA kit, according to manufacturer instructions. RNA-Seq was performed at the Caltech Millard and Muriel Jacobs Genetics and Genomics Laboratory at 35 million reads on two biological replicates for both the control cranial and Draxin overexpression cranial neural crest cells. Sequencing libraries were built according to Illumina Standard Protocols and SR50 sequencing was performed in a HiSeq Illumina machine by the Caltech Millard and Muriel Jacobs Genetics and Genomics Laboratory. Sequence reads were aligned to the G. gallus genome (galgal6) with Bowtie2 (Langmead and Salzberg, 2012 (link)), transcript counts were calculated with HTSeq-Count (Anders et al., 2015 (link)), and differential expression analysis was performed with DESeq2 (Love et al., 2014 (link)). Gene lists were analyzed for functional annotation using PANTHER (Mi et al., 2019 (link)) and DAVID (Huang da et al., 2009a (link),b (link)).