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  • br Experimental design materials and methods br Data The

    2018-11-03


    Experimental design, materials and methods
    Data The data reported include supporting information to the phylogenetic analyses of Franchi et al. [1]. They consist of transcript sequences, sequence alignments and comparisons of four apoptosis-related genes identified in the recently-obtained transcriptome of B. schlosseri[2]. The sequences show high similarity with mammalian transcripts for Bax, AIF1, PARP1 and IAP7 and were named BsBax, BsAIF1, BsPARP1 and BsIAP7, respectively. The expression of these genes was studied further in the above-reported paper [1].
    Experimental design, materials and methods Amplification and cloning of transcripts for BsBax, BsAIF1, BsPARP1 and BsIAP7 was achieved with specific primers designed on sequences found in our collection of transcriptomes [2]. In order to verify and complete the full length cDNA, PCR reactions were carried out with a denaturing step at 94°C for 2min, 40 cycles of 30s at 94°C, 40s at 60°C and 90s at 72°C, and a final extension at 72°C for 10min. Amplicons were separated using 1.5% agarose gel, purified, cloned and sequenced. The partial transcripts were elongated through 5′ and 3′ RACE according to the 2nd generation of 5′/3′ RACE kit (Roche). Supplementary Table 1 reports the specific primers used for amplicons production and their elongation through 5′- and 3′-RACE and for the in situ hybridisation experiments reported in [1]. The sequences from GenBank, reported in Supplementary Tables 2–5, were used for alignments and sequence comparisons with the sequences of BsBax, BsAIF1, BsPARP1, BsIAP7, respectively. The latter were deposited in GenBank and corresponding to the accession numbers GenBank: KU948200, GenBank: KU948201, GenBank: KU948202 and GenBank: KU948203, respectively (Figs. 1–8). The predicted amino HG-9-91-01 sequences of BsBax, BsAIF1, BsPARP1, BsIAP7 are reported in Figs. 2, 4, 6 and 8, respectively. They were aligned with known orthologous sequences from both vertebrate and invertebrates using the MUSCLE programme [3] and data are reported in Figs. 1, 3, 5, and 7, respectively. Identity analysis of the deduced amino acid sequences were performed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and LALIGN (http://www.ch.embnet.org/software/LALIGN_form.html) [4] and are reported in Supplementary Tables 2–5.
    Acknowledgements This research was supported by grants from the CARIPARO Foundation (Excellence Project 2009) and the Italian M.I.U.R (P.R.I.N. grant 20109XZEPR to LB).
    Specifications Table Value of the data
    1. Data This data consist of five high-throughput sequenced samples of barley roots (Supplementary Table 1, n=2) and upper part (Supplementary Table 2, n=3), exposed to optimal or drought conditions and subsequent re-watering, generated from an Illumina HiSeq 2500, together with GO term analysis of the most affected Biological Processes (Tables 1–3). Predicted genes from the latest genome version (082214v1.25) have been annotated based on three various databases (Fig. 1) and associated to GO term categories (Fig. 2). Several GO terms have been assigned to each predicted sequence (Fig. 3).
    Experimental design, materials and methods
    Acknowledgments This work was supported by the Czech Science Foundation (Grant no. 14-12355 S) and the National Program for Sustainability (Project no. LO1204).
    Data The pRSET expression vector encoding a putative protein containing 220 amino acids was constructed (Fig. 1A). The protein in Escherichia coli was purified using a 1st Ni-NTA Sepharose column and a 2nd Sepharose column (Fig. 1B,C). After mice were immunized with the antigen, the supernatants of the hybridoma cells were analyzed by using indirect ELISA (Fig. 2). To establish a sandwich-ELISA system, the intersection method was used with HRP-labeled antibodies (Table 1). The quantities of rec-FSHβ/α and luteinizing hormone (LH) β/α and the selected stable cell lines, and western blot result were described (Fig. 3). The FSHβ-subunit antibody (eFB-C14) was used for examining FSH localization in the pituitary during oocyte maturation (Fig. 4).