R2 - Horizontal Anaerobic Reactor
Total genome DNA from samples was extracted using CTAB/SDS method. DNA concentration and purity was monitored on 1% agarose gels. According to the concentration, DNA was diluted to 1ng/L using sterile water.Amplicon Generation16S rRNA/18SrRNA/ITS genes of distinct regions (16SV4/16SV3/16SV3-V4/16SV4-V5, 18S V4/18S V9, ITS1/ITS2, Arc V4) were amplified used specific primer (e.g. 16S V4: 515F-806R, 18S V4: 528F-706R, 18S V9: 1380F-1510R, et. al ) with the barcode. All PCR reactions were carried out with Phusion High-Fidelity PCR Master Mix (New England Biolabs).PCR Products quantification and qualificationMix same volume of 1X loading buffer (contained SYB green) with PCR products and operate electrophoresis on 2% agarose gel for detection. Samples with bright main strip between 400bp-450bp were chosen for further experiments.PCR Products Mixing and PurificationPCR products was mixed at equal density ratios. The mixed PCR products were purified with Qiagen Gel Extraction Kit (Qiagen, Germany).The libraries generated with NEBNext UltraTM DNA Library Prep Kit for Illumina and quantified via Qubit and Q-PCR, would be analysed by Illumina platform.