title: nifH Gene Sequencing of Sphagnum multifibrosumcapitula
design: Sequencing libraries were constructed using nifH gene amplicons. Total DNA was extracted from the same batch of frozen samples (consistent with the 16S rRNA gene sequencing experiment). PCR was performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs) to amplify the nifH gene using primers PolF (5-TGCGAYCCSAARGCBGACTC-3) and PolR (5-ATSGCCATCATYRCCGGA-3) (Poly et al., 2001). The PCR protocol consisted of an initial denaturation at 98C for 2 minutes, followed by 30 cycles of denaturation at 98C for 15 seconds, annealing at 55C for 30 seconds, and extension at 72C for 30 seconds, with a final extension at 72C for 5 minutes and hold at 10C. PCR products were verified by 2% agarose gel electrophoresis and purified using AXYGEN Gel Extraction Kit. Purified products were quantified via Microplate reader FLx800 (BioTek) with Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific). Final libraries were prepared for paired-end sequencing (2300 bp) on the Illumina MiSeq platform, using MiSeq Reagent Kit V3 (601 cycles).