title: NextSeq 500 sequencing; RNA-seq of GFP positive and negative fractions of zebrafish transgenic cell lines of sea sponge enhancers
design: RNA-seq of GFP positive and negative fractions of zebrafish transgenic cell lines of sea sponge enhancers
library_construction_protocol: 72hpf zebrafish embryos were anesthetised with tricane diluted 1:1 in the embryo medium and the yolks were mechanically removed by pipetting up and down at least 10 times in calcium free ringer's solution. Zebrafish embryos were further digested in PBS containing 0.25% liberase at 28°C for 5 minutes and a single cell solution was prepared by passing through a 40μm nylon mesh. Both GFP positive and negative single cells were sorted into Trizol LS reagent (Life Technologies), respectively with a BD FACSAria Cell Sorter (BD Biosciences).  RNA was extracted using a Direct-zol RNA extraction kit (Zymo research). RNA-Seq libraries were prepared from purified total RNA using a modified Smart-Seq2 protocol developed by Picelli et al. 55. 2 ng of purified total RNA (0.4 ng/µL) was combined with 1 µL of 10 µM oligo-dT primer (/5Biosg/AAGCAGTGGTATCAACGCAGAGTACT30VN; Integrated DNA Technologies) and 1 µL of dNTP mix (10 mM each; Invitrogen, y02256), then the protocol was continued as described (ref. 2). Briefly, the RNA was reverse transcribed with the Smart-Seq2 TSO (/5Biosg/AAGCAGTGGTATCAACGCAGAGTACATrGrGrG, Integrated DNA Technologies), followed by 12 cycles of PCR amplification to obtain enough cDNA to prepare a library. Volumes of reagents were scaled accordingly to maintain final concentration ratios as in the original protocol, except for the PCR preamplification where the Smart-Seq2 ISPCR primer (/5Biosg/AAGCAGTGGTATCAACGCAGAGT; Integrated DNA Technologies) was added to a final concentration of 0.25 µM. 0.5 ng of cDNA was prepped into a library using the Nextera XT DNA Library Prep Kit (Illumina, FC-131-1096), with 12 cycles of PCR used to amplify the final library. The final Nextera XT libraries were quantified on the Perkin Elmer LabChip GX with the DNA High Sensitivity Reagent kit (Perkin Elmer, CLS760672). Libraries were pooled in equimolar ratios.