title: Full transcriptome analysis of early dorsoventral (DV) patterning in zebrafish- Bcat
design: RNA quality and quantity measurements were performed on Bioanalyzer (Agilent Technologies) and Qubit (Life Technologies). High quality (RIN >8.5) total RNA samples from three biological replicates were pooled and processed using the SOLiD total RNA-Seq Kit (Life Technologies), according to the manufacturers suggestions. Briefly, 5mg of pooled RNA was DNaseI treated and the ribosomal RNA depleted using Eucaryote RiboMinues rRNA Removal Kit (Life Technologies). The leftover was fragmented using RNaseIII, the 50-200nt fraction size selected, sequencing adaptors ligated and the templates reverse transcribed using ArrayScript RT. The cDNA library was purified with Qiagen MinElute PCR Purification Kit (Qiagen) and size-selected on a 6% TBE-Urea denaturing polyacrylamide gel. The 150-250nt cDNA fraction was amplified using AmpliTaq polymerase and purified by AmPureXP Beads (Agencourt). Concentration of each library was determined using the SOLiD Library TaqMan Quantitation Kit (Life Technologies). Each library was clonally amplified on SOLiD P1 DNA Beads by emulsion PCR (ePCR). Emulsions were broken with butanol, and ePCR beads enriched for template-positive beads by hybridization with magnetic enrichment beads. Template-enriched beads were extended at the 3' end in the presence of terminal transferase and 3' bead linker. Beads with the clonally amplified DNA were deposited onto sequencing slide and sequenced on SOLiD V4 Instrument using the 50-base sequencing chemistry.