Fastdigest rsai

The 2^nd–11^th fractions of each sample (10 samples; total 100 fractions) were analyzed with T-RFLP fingerprinting as described by Zhang et al. [33 (link)]. Briefly, the fluorescently labeled forward primer 27F (5' [6FAM]-AGAGTTTGATCMTGGCTCAG-3') and the unlabeled reverse primer 927R (5'-ACCGCTTGTGCGGGCCC-3') were used to amplify bacterial 16S rRNA genes [34 , 35 (link)]. PCR products were checked by electrophoresis on 1% agarose gel and purified with an agarose gel DNA purification kit (Tiangen Biotech Co., Ltd., Beijing, China). Purified products were digested with restriction enzyme FastDigest RsaI (Thermo Fisher Scientific, Inc.) at 37°C for 1–2 h. The digested products were recovered using 20 uL of sterile deionized water and ethanol precipitation. Purified products were then mixed with 0.5 uL of an internal size standard (ET ROX-900) and then detected using a MegaBACE genetic analyzer (Amersham Biosciences Corp., Piscataway, NJ, USA). The output was transferred to T-REX software (http://trex.biohpc.org/) [33 (link), 36 (link)] for noise removal and construction of a data matrix. The obtained matrix was further analyzed with Primer 5 analysis software to determine fragment profiles of the 12 density gradient fractions from each experimental sample as well as community similarity between fractions. Typical ¹³C- and ¹²C-fractions were chosen for pyrosequencing.