Exoprostar 1 step

Symbiodiniaceae communities were characterized using next‐generation sequencing of the rDNA ITS2 region (ITS2). Triplicates for each sample were pooled, and samples were cleaned using ExoProStar 1‐step (GE Healthcare). Samples were then indexed using the Nextera XT Index Kit v2 (dual indexes and Illumina sequencing adaptors added). Successful addition of indexes was confirmed by comparing the length of the initial PCR product to the corresponding indexed sample on a 1% agarose gel. Samples were then cleaned and normalized using the SequalPrep Normalization Plate Kit (Invitrogen). The ITS2 libraries were pooled in an Eppendorf tube (4 μl per sample) and concentrated using a CentriVap Benchtop Vacuum Concentrator (Labconco). Following this, quality of the library was assessed using the Agilent High Sensitivity DNA Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies). Quantification was done using Qubit (Qubit dsDNA High Sensitivity Assay Kit; Invitrogen). Sequencing was performed at 6 pM with 20% phiX on the Illumina MiSeq platform at 2 × 301 bp paired‐end in the KAUST Bioscience Core Laboratory (BCL). Raw sequencing data are available under NCBI BioProject accession number #657469.

Sequencing of SNPs in Lbx1 (P52955) and Tlx1 (P43345) was performed to clarify whether SNPs in background strains A (for A.SW) and C57BL/6 (for B10.S), according to the Ensembl and MGI databases, were present in A.SW and B10.S mice because dHPLC did not show any SNPs. PCR primers covering exons in which SNPs were predicted, including exon/intron borders, were used to generate PCR products (see Table S4). Residual nucleotides were removed using ExoProStar 1-Step (GE Healthcare), and the PCR products were sequenced according to a standard protocol for fluorescently labeled dideoxynucleotides (Applied Biosystems, Life Technologies) and separated on a capillary electrophoresis instrument (ABI 3500, Life Technologies).

The purification of PCR products was performed using ExoProStar 1-Step (GE Healthcare, Chicago, IL, USA), as instructed by the manufacturer. Bidirectional Sanger sequencing was accomplished using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher) and the sequences generated in ABI-3500 (Applied Biosystems, Foster City, CA, USA). The sequences generated here were deposited in GenBank under accession numbers OP762196 to OP762335.