Neonatal and Adult Isl-1+ CPC RNA Sequencing

RNA samples isolated from neonatal and adult Isl-1+ CPC clones were sent to PrimBio Research Institute for RNA-Sequencing (Exton, PA, USA). rRNA was removed from the total RNA sample using an rRNA removal kit from Illumina (San Diego, CA, USA) (cat# MRZG12324). Sequencing libraries were assembled with an Ion Total RNA-Seq Kit v2 from Thermo Fisher (Waltham, MA, USA) (Cat# 4479789). Nucleic acid binding beads from Ambion (Austin, TX, USA) were used to purify the cDNA library (Cat# 4479681) prior to PCR amplification. Agilent dsDNA High Sensitivity kit was used to determine the quality of the library (Agilent, Santa Clara, CA, USA). The samples were enriched via an Ion OneTouch ES instrument and an Ion PI™ Hi-Q™ OT2 200 Kit (Thermo Fisher, Waltham, MA, USA). Sequencing was performed using an Ion Proton sequencer (Thermo Fisher, Waltham, MA, USA) and a species-specific protocol for our samples. Next, sequence files were aligned to the human genome and quality was determined using the Strand NGS program. Normalization and quantification of the aligned reads were performed using the Deseq algorithm within the Strand NGS program. The Audic–Claverie test and the Benjamini–Hochberg correction test were used for statistical analysis. Significance was determined using a 2.0-fold change minimum cutoff.