Nextera library prep kit

A modified version of the Omni-ATAC protocol was used to generate ATAC-seq libraries [20 (link),21 (link)]. After cells were collected from FACS, 50,000 cells per sample were spun down at 500× g for 7 min at 4 °C. Cells were then resuspended in 500 µL cold solution comprised of 50% FBS, 40% DPBS, 10% DMSO. Cells were cryopreserved at -80 °C overnight. Prior to the ATAC-seq assay, cells were thawed in a 37 °C water bath, pelleted, washed with cold PBS, and tagmented as described in Buenrostro et al. (2013) with modifications [20 (link),21 (link)]. Briefly, cell pellets were resuspended in lysis buffer, pelleted, and tagmented using the enzyme and buffer provided in the Nextera Library Prep Kit (Illumina, San Diego, CA, USA). Tagmented DNA was then purified using the MinElute PCR purification kit (Qiagen, Hilden, Germany), amplified with 10 cycles of PCR, and purified using Agencourt AMPure SPRI beads (Beckman Coulter, Brea, CA, USA). The resulting material was quantified using the KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems, Wilmington, MA, USA) and sequenced with PE42 sequencing on the NextSeq 500 sequencer (Illumina, San Diego, CA, USA).

Sequencing libraries were prepared using the Illumina Nextera library prep kit with 50 ng of input DNA and sequenced on the Illumina MiSeq machine (2 × 300 bp). Genome assembly was performed using Newbler (Margulies et al., 2005 (link)) and manually inspected using Consed (Gordon and Green, 2013 (link)). Annotation was performed automatically with prokka (Seemann, 2014 (link)) and refined with manual annotations from BLASTp homologies using GenDB (Meyer et al., 2003 (link)).

The cryopreserved jejunum was sent to Active Motif for ATAC-seq experiments. The nuclei isolated from the tissue were tagmented (fragmented and tagged with sequencing adaptors) by hyperactive Tn5 transposase as described previously^{72 (link)} with some modifications^{73 (link)} using the reagents in Nextera Library Prep Kit (Illumina). After amplification with ten cycles of PCR, the resultant DNA was sequenced with PE42 sequencing on the NextSeq 500 sequencer (Illumin). Sequence reads were aligned using the BWA algorithm,^{74 (link)} and peaks in the histograms were identified using the MACS 2.1.0 algorithm at a cutoff of p value 1 × 10⁷. Signal maps and peak locations were analyzed using Active Motifs proprietary analysis program, and reads counted in all merged peak regions were compared using DeSeq2.^{75 (link)}

We extracted genomic DNA, from fin clips of 22 male individuals^{62 (link)} (Qiagen DNeasy, Cat #69504), from 8 rock dwelling and 14 sand dwelling Lake Malawi species (Supplementary Table S2) representing broad diversity across the rock and sand lineages in Lake Malawi. We made libraries using the Illumina Nextera Library prep kit and performed paired-end sequencing on the Illumina Hi-Seq 2500 at Georgia Tech. The Metriaclima zebra reference genome version MZ_UMD2a^{34 (link)} was used for genome alignment, variant discovery and annotation using standard BWA^{63 (link)} and GATK practices^{64 (link)}. The maximum likelihood tree in Fig. 1A was constructed using SNPhylo^{65 (link)}, from variant data.

The ATAC-Seq was performed by Active Motif as described by Buenrostro et al. [32 (link)], with some modifications based on Corces et al. [52 (link)]. Briefly, cell pellets were resuspended in lysis buffer, pelleted, and tagmented using the enzyme and buffer provided in the Nextera Library Prep Kit (Illumina, Inc.). Tagmented DNA was then purified using the MinElute PCR purification kit (Qiagen), amplified with 10 cycles of PCR, and purified. The ATAC-Seq libraries were sequenced as 45 bp paired-end libraries on a NextSeq 500 and mapped to the human (version hg19) or mouse (version mm10) genomes using BWA [53 (link)] with default parameters. Reads were filtered using Illumina’s sequence quality filters, and PCR duplicates were removed. Peaks were called using MACS1.4.2 at a cutoff of p-value = 1e-7, without control file, and with the–nomodel option [54 (link)]. Peaks were merged for all samples of the same background, reads were counted for each region, and differential analysis was performed between each modified sample and its parent strain using DESeq2 [37 (link)].

ATAC-seq of four samples (two BW and two AW) was performed by Active Motif, Inc. (Carlsbad, CA, USA). Before the sequencing, a 50 mL digestion solution (1% trypsin and 1.15 mmol CaCl₂ in phosphate-buffered saline) was added to the rumen epithelial tissue and incubated at 37 °C for 15 min to dissociate the cells. Rumen epithelial fragments underwent five to six cycles of digestion with trypsin solution. The first two rounds of digestion solution were discarded, and the third, fourth, and fifth rounds were collected and combined. The cell samples were thawed in a 37 °C water bath, pelleted, and washed with cold PBS. The resulted cells were tagmented as previously described [11 (link),16 (link)]. In brief, cell pellets were resuspended with lysis buffer, pelleted, and tagmented using the enzyme and buffer provided in the Nextera Library Prep Kit (Illumina, San Diego, CA, USA). The MinElute PCR purification kit (QIAGEN, Germantown, MD, USA) was used to purify tagmented DNA, and then, the DNA was amplified with 10 cycles of PCR and purified using Agencourt AMPure SPRI beads (Beckman Coulter, Pasadena, CA, USA). The resulting material was quantified using the KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems, Wilmington, MA, USA). The DNA libraries were sequenced (2 × 75 bp) on a HiSeq 2500 platform (Illumina, San Diego, CA, USA).