Tapestation d1000 kit

RNA sample with a RIN value above seven was used for cDNA synthesis. Equal amounts of high-quality RNA from accessory gland tissues of virgin males and males interrupted during mating were taken for cDNA synthesis. The cDNA library construction was done using NEB Next Ultra RNA Library Prep Kit and Illumina sequencing of the samples was performed at Sandor Life Sciences Pvt. Ltd. (Hyderabad, India). The mRNA was purified from 1 μg of the total RNA using Oligo dT beads and was fragmented into short sequences at the appropriate temperature. These fragments served as templates and the first strand of cDNA was synthesized using random primers and reverse transcriptase, followed by the synthesis of the second-strand cDNA using RNaseH and DNA polymerase I. After end-repair, the fragments were ligated using sequencing adaptors and purified using AMPure XP beads. These adaptor-ligated fragments were amplified using PCR and the products were further purified using AMPure XP beads to create a cDNA library and were assessed on the Agilent Bioanalyzer 2100 system. Quantification and size distribution of the prepared library was determined using a Qubit flourometer and Agilent Tape station D1000 Kit according to the manufacturer’s instructions. The resulting cDNA libraries were then paired-end sequenced (2 × 150 bp) with the Illumina HiSeq^TM platform.

DNA was extracted as previously reported^{56 (link)}. Briefly, DNA was quantified with Quant-iT dsDNA Assay kits (Invitrogen, ThermoFisher, USA), and normalized to 50ng DNA. MGIEasy FS DNA Library Prep Set kit (MGI, Shenzhen, China) was used to prepare DNA libraries. Quality control of all libraries was done by measuring their concentration with Quant-iT dsDNA Assay kits and fragment size with TapeStation D1000 kit (Agilent, USA). Libraries were pooled with an equal amount of DNA from each sample. Circularized DNA libraries were sequenced using 100 bp paired-end sequencing on a DNBSEQ-T7 sequencer (MGI, Shenzhen, China). Positive (ZymoBIOMICS Community standard, Zymo Research, Irvine, CA, USA, Supplementary Table 6) and negative (DNA/RNA shield) controls were included for both extraction and sequencing steps.

All libraries were quantified with qPCR using the NEBnext Library Quant Kit for Illumina and checked for fragment size using the TapeStation D1000 kit (Agilent). The libraries were pooled in equimolar concentration for a total pooled concentration of 2 nM. 10x single-cell libraries were sequenced using the Illumina NovaSeq 6000 and S2 flow cells (100 cycle kit) with a read one length of 26 cycles, and a read two length of 92 or 98 cycles for version 2 chemistry. Version 3 chemistry had a read one length of 28 cycles, and a read two length of 94 cycles. Bulk libraries were sequenced on the Illumina NovaSeq 6000 using SP flow cells (100 cycle kit) with read length of 150 for dissociated bulk in C57BL/6J mice, 51 for undissociated bulk in Balb/c male mice, and 60 for Balb/c female mice.