Mock: Male: 2
Libraries for next-generation sequencing were prepared using the NEBNext Ultra RNA Library Prep kit (NEB #E7530L, MA, USA) according to the manufacturers instructions using NEBNext Mutliplex Oligos for Illumina Index primer sets #1 and #4. Information regarding specific samples and their indices may be found in the Supplemental Data accompanying this manuscript. Libraries were quantified by Qubit Flex Fluorometry using the Qubit 1X dsDNA HS Assay Kit (ThermoFisher, Cat. No. Q33231, MA, USA). The average Fragment length of each sample was assessed using an Agilent Fragment Analyzer 5200, utilizing either the Standard Sensitivity Next Generation Sequencing kit, or the High Sensitivity Next Generation Sequencing Kit, depending on the mass as determined by the Qubit assay. Equimolar amounts of each sample were pooled, and the mass of the pool was quantified by Qubit using a 1X dsDNA HS assay kit. The Qubit concentration reading, and the average fragment length were used to determine molar concentration for loading. The library and PhiX standards were diluted using the standard normalization method following the manufacturer's directions. The total volume of the library was 20 L at 750 pM, with 2% PhiX spike in. Further denaturing and dilution of the library to 75pM is done on the instrument (NextSeq 2000). Sequencing was performed on a NextSeq 2000 using a P3 200-cycle reagent kit with a P3 flow cell.