Bluepippin instrument

Adapter-dimer removal was performed using the BluePippin instrument (Sage Science, Beverly, MA, USA) with 3% agarose cassettes and internal standards, to automatically separate the miRNA sequencing library from their adapter dimers. The BluePippin optical system was calibrated and the continuity test was completed before every run. The size selection was performed according to the manufacturer’s protocol, with the size-selection mode set to tight 180 bp. After adapter-dimer removal, the cleaned miRNA-sequencing library was subjected to the Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA) for pre sequencing quantification/quality control. Therefore, 1 µL of each miRNA sequencing library was analyzed using a High Sensitivity DNA chip according to the manufacturer’s instructions.

The genomic DNA of Tanzania and Beauregard were extracted using the method cetyltrimethyl ammonium bromide and purified with 1× Agencourt AMPure XP beads (Beckman Coulter), according to manufacturer’s instructions. Before the library preparation, 1.5 µg purified gDNA was size selected using the BluePippin instrument (Sage Science) with the 0.75% Agarose Dye free, Marker U1 High-pass 30–40 kb vs3 protocol followed by a purification step with 0.4× AMPure XP beads. The library preparations for these two samples were done following the Chromium
^TM Genome Reagent Kits user guide (CG00022, Rev C). In summary, 10 ng of sample DNA was used to generate Gel Bead-In-Emulsions (GEM) in the Chromium
^TM Controller (10× Genomics) followed by isothermal incubation, post GEM incubation cleanup and quality control (QC). Libraries were constructed with end-repair and A-tailing, adaptor ligation, post ligation cleanup using SPRIselect Reagent (Beckman Coulter, USA), sample index PCR, post PCR cleanup, and QC. We modified the protocol by increasing the number of PCR cycles to nice and adding 105 µl SPRIselect Reagent for the Post Sample Index PCR Cleanup, which resulted in the recovery of shorter fragments than it was expected. The libraries were sequenced using the HiSeq X Ten platform (Illumina, San Diego, CA).

Sequencing was performed at the University of Arizona Genetics Core following previously described methods [31 (link)]. Briefly, 2 μg of DNA was sheared to 500 bp by ultrasonication on a Covaris S2. Samples were end-repaired, A tailed and ligated to adaptors Ind_Ad_T and Ind_Ad_B by NEBnext DNA library prep kit. PCR enrichment of TN5 junctions was performed with primers Tn-FO and Adapt-RO using Kapa HotStart HiFi (Roche) using thermocycling conditions as follows; 95° C for 5 minutes, 94° C for 45 seconds, 56° C for 1 minute, 72° C for 1 minute for 22 cycles, and a final extension at 72° C for 10 minutes. Samples were purified using a MagBio HighPrep PCR cleanup kit (MagBio Genomics) and amplicons from 100 to 600 bp were captured using a 2% agarose cassette on a Blue Pippin instrument (Sage Science). Amplicons were indexed using a Nextera V2 dual index kit (Illumina) and purified by MagBio HighPrep PCR cleanup. Purified, indexed amplicons were then quantified on a Qbit fluorometer, and sequenced on an Illumina MiSeq instrument as 75 bp paired end reads. Sequencing was repeated on these libraries in 3 separate runs to generate enough reads for analysis, and the reads were merged by sample after initial demultiplexing.