GSM6260326: E13.5 Esophagus Rep 2 [13-5E2]; Mus musculus; RNA-Seq
RNeasy Micro Kit 50 (Qiagen, Valencia, CA) was used for extraction of total RNA according to the manufacturer's instructions. DNA digestion was performed using the DNase enzyme eliminator column- RNeasy MinElute spin columns. RNA concentration yield and integrity were evaluated using a Nanodrop ND-1000 spectrophotometer (Nanodrop, Wilmington, DE), and samples that met the following three criteria were retained: a concentration of 1µg, an OD260−280 of 1.8–2.0 and an OD260:230 above two. Additionally, samples were evaluated for RNA integrity value (RIN) using the Agilent 2100 Bioanalyzer and RNA 6000 Pico kit (Agilent, Santa Clara, CA) according to the manufacturer's instructions. Only samples with electropherograms exhibiting distinct 18S and 28S ribosomal RNA (rRNA) peaks and no evidence of degradation were retained. Poly-A library preparation and RNA sequencing was performed by the DNA Sequencing facility at the University of Wisconsin-Madison Biotechnology Center per standard protocols. Briefly, first-strand cDNA using random hexamer-primed reverse transcription was generated, followed by second-strand cDNA synthesis using RNase MODULE and DNA polymerase, and ligation of sequencing adapters using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA). Fragments of 350 bp were selected by gel electrophoresis, followed by 15 cycles of PCR amplification. Sequencing of the prepared libraries was done on Illumina's HiSeq 2000 and Illumina NovaSeq6000 2x150 S4 platforms with four RNA-Seq libraries per lane with a minimum depth of 2 × 100 bp paired-end reads by the University of Wisconsin-Madison DNA Sequencing Facility in the University of Wisconsin-Madison Biotechnology Center.