title: RNA-Seq of red mud
design: Extracted DNA was diluted to 10 ng/mL and stored at 20 C for downstream use. Two-round PCR amplification was conducted with primers 515F (5-GTGCCAGCMGCCGCGGTAA-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3) designed to be universal for bacteria and archaea. In the first-round PCR, each 25 ml reaction volume contained 1 ml of each 10 mM primer, 10 ng of template DNA, and 0.5 units of Accuprime high-fidelity Taq (Invitrogen, USA). The PCR procedure was: 94 C for 1 min; 10 and 20 cycles were used for the 1st and 2nd round, respectively, at 94 C for 20 s, 53 C for 25 s, and 68 C for 45 s; 68 C for 10 min. Each sample was amplified with three technical replicates, and then they were mixed in one tube, purified with a gel extraction kit (QIAGEN, USA) and used as a template for the second-round PCR. In the second PCR reaction, the reaction condition was the same as that in the first-round PCR except that barcoded primers were used. After two-round amplification, PCR products from three technical replicates were quantified with electrophoresis and mixed in one tube. All samples were pooled together with equal molar amounts from each sample and purified through the QIAGEN gel extraction kit. The purified library was diluted, denatured, re-diluted, mixed with PhiX, as described in the Illumina library preparation protocols, and then applied to an Illumina Miseq system for sequencing with the Reagent Kit v2 2250 bp.