Agilent 21100 bioanalyzer

Embryos from the same litters were pooled and used as individual biological replicates. Total RNA was isolated from FACS purified basal epidermal cells, (see above for a description of the purification strategy) using Direct-zol RNA MiniPrep kit (Zymo Research) per manufacturers instruction. RNA quality was determined using Agilent 21100 Bioanalyzer and all samples had a RIN >0.8. Poly-A enrichment and library preparation using Illumina TruSeq mRNA sample preparation kit was performed by Weill Cornell Medical College Genomic Core Facility. Samples were sequenced on Illumina HiSeq4000 and 50 bp Single-end reads obtained. Transcripts were quantified to generate TPM and counts using Salmon (Patro et al., 2017 (link)) with Gencode M18 (mm10) used as the reference. Differentially expressed genes were determined using DESeq2 (Love et al., 2014 (link)) with lfcThreshold = 0 and alpha = 0.01 contrasting shRhou and shScr.

Total RNA was extracted using a modified CTAB method55 (link) and was evaluated using an Agilent 21100 Bioanalyzer (Agilent Technologies). Small RNA libraries were prepared using the standard protocols of the Illumina Small RNA Sample Prep Kit and were sequenced using an Illumina Genome Analyzer (Illumina, San Diego, CA, USA) at BGI (Shenzhen, China).