16S rRNA gene of lake microbiome: L227 winter 9.5m C
Lake water was filtered through Sterivex or Stericup filters (0.22 m pore size, Millipore). Filters were removed from the filtration capsule or cup using sterile scalpel blades, placed in DNA/RNA Shield solution (Zymo Research), and stored at 4C overnight for sample preservation. Samples were then stored at 80C until nucleic acid extraction with PowerWater RNeasy extraction kit (Qiagen), skipping the step for DNA digestion to recover both DNA and RNA from the same filter. Quality of the extracted nucleic acids was verified by gel electrophoresis, and PCR amplification of the 16S rRNA gene with primers described below was carried out in every sample to check for PCR inhibition.PCR primers 515F-Parada (5-GTGYCAGCMGCCGCGGTAA-3) and 926R-Quince (5-CCGYCAATTYMTTTRAGTTT-3) were used for amplification (Parada et al., 2016, Quince et al. 2011). 16S rRNA gene amplicons were sequenced in a paired-end approach using the MiSeq System (Illumina).
SUB15810121
L179C