Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Total RNA was extracted using RNeasy Micro kit (Qiagen, Cat# 74004) as per manufacturer's instructions, including on-column DNase I digestion 15 mins at room temp to eliminate genomic DNA. Extracted Total RNA from anterior halves of NF stage 15 embryos was verified on the Agilent Bioanalyzer system using a nanochip to verify the RNA integrity number (RIN). High quality total RNA (500ng) was input into the Illumina Truseq v2 stranded mRNA library prep kit (cat#20020595) according to the manufacturer's protocol. Generated cDNA was amplified with 12 pcr cycles. Final generated libraries were verified on the Agilent Tapestation 2200 system using high sensitivity DNA Screentape. Generated cDNA was amplified with 12 pcr cycles. Final generated libraries were verified on the Agilent Tapestation 2200 system using high sensitivity DNA Screentape. Sample concentration was checked on the Invitrogen qubit system using the high sensitivity DNA reagent. Each sample was normalized to a specific nanomolar concentration and pooled equally into a final combined pool. The pool was run on 1 lane of Novaseq SP100 flow cell (run length was 50 paired end bases) on the Illumina Novaseq 6000 sequencing system.