title: RNA-seq green roof substrate
design: Total microbial genomic DNA was extracted from the collected substrate samples using the E.Z.N.A. Soil DNA Kit (Omega Bio-tek Inc., the U.S.) according to manufacturers instructions. The quality and concentration of DNA were determined by 1.0% agarose gel electrophoresis and the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., the U.S.) and kept at -80  prior to further use. The hypervariable region V3-V4 of the bacterial 16S rRNA gene and the fungal ITS rRNA were respectively amplified with primer pairs 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'), ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2R(5'-GCTGCGTTCTTCATCGATGC-3') by the T100 Thermal Cycler (Bio-Rad Laboratories, Inc., the U.S.). The PCR reaction mixture including 4 L 5  Fast Pfu buffer, 2 L dNTPs (2.5 mM), 0.8 L each primer (5 M), 0.4 L Fast Pfu polymerase, 10 ng of template DNA, and ddH2O to a final volume of 20 L. PCR amplification cycling conditions were as follows: initial denaturation at 95  for 3 min, followed by 27 cycles of denaturing at 95  for 30 s, annealing at 55  for 30 s and extension at 72  for 45 s, and single extension at 72  for 10 min, and end at 4 . The PCR product was extracted from 2% agarose gel and purified using the AxyPrep PCR Clean-Up Kit (Axygen Biosciences, Inc., the U.S.) according to manufacturers instructions and quantified using the Qubit 4.0 fluorometer (Thermo Fisher Scientific Inc., the U.S.).