D1000 screentape reagents

We performed 16S rRNA sequencing of bacterial DNA isolated from menstrual cup cell pellets according to Illumina’s 16S metagenomics sequencing library preparation guide using the MiSeq Reagent Kit v3 (600-cycle) and MiSeq sequencing platform according to the manufacturer’s instructions (https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany). Prior to DNA isolation, cell pellets were homogenized in approximately 200 µL of PBS using pre-filled silica bead tubes (VWR, Radnor, Pennsylvania) and a Bead Ruptor Elite homogenizer (OMNI International, Kennesaw, Georgia) using the following settings: 3 cycles of bead beating at 6 m/s for 45 seconds followed by a 2 min dwell period. PCR amplification verification and library validation was done using a TapeStation electrophoresis system and D1000 ScreenTape reagents (Agilent, Santa Clara, California). Libraries were quantified using a Qubit 4 Fluorometer and the Qubit dsDNA HS assay kit (ThermoFisher Scientific, Waltham, Massachusetts). The QIIME2 platform was used to process sequence data, perform quality control, and generate a table representing the number of reads that mapped to specific operational taxonomic units (OTUs).

PCR products were diluted to 20 uL with PCR grade water and cleaned up using 1.0X AMPureAP beads (Beckman Coulter, Brea, CA), vacuum-dried, reconstituted in 12 uL of PCR grade water, quantified using a Quant-It dsDNA HS assay kit (Thermo Fisher Scientific Inc., Waltham, MA), normalized and pooled. The sequencing pool was concentrated, cleaned up using 1.8X AMPureAP beads (Beckman Coulter, Brea, CA) quantified using a Quant-It dsDNA HS assay kit (Thermo Fisher Scientific Inc., Waltham, MA). Sequence pool was assessed for purity and the presence of 725 bp peak (±20%) using a 2200 TapeStation system and D1000 Screen tape/reagents (Agilent Technologies, Santa Clara, CA).