Xu Lab, School of Life Sciences, Sun Yat-sen University
SAPAS sequencing library was constructed as described in Fu et al. 2011. Total RNA was extracted from zebrafish embryos by TRIzol, and ∼10 μg of total RNA was randomly fragmented by heating. An anchored oligo d(T) primer and a 5′ template switching linker tagged with Illumina adaptors were used in template switch reverse transcription (RT) by SuperScript II reverse transcriptase from Invitrogen. Two mutations in the poly(A) were introduced by PCR amplification with a determined number of cycles to ensure that the ds cDNA remain in the exponential phase of amplification. The PCR products were recovered after PAGE. The size-selection of 250–400 bp was performed by PAGE gel-excision. The recovery was quantified by a Qubit 2.0 Fluoromete, and the average size was determined by Agilent 2100 bioanalyzer. A quality control was performed by plasmid recombinant and Sanger sequencing. The recovery was ligated to pGEM-T Easy Vector and transformed into DH5а competent cells. Plasmid DNA was extracted and sequenced by ABI 3730 DNA Analyzer. Each end of the insertion sequence should be the Illumina sequence primer. The insertion sequence with long poly(A) stretch should be <5%, and most of the insertion sequence should be mapped to the zebrafish genome.