Moraine Bacteria sample 4
The top 12 cm of soil was first removed using an aseptic metallic shovel previously cleaned with 70% ethanol. Then, 300 g of soil samples were taken for each biological replica from the soil underneath (up to 10 cm), stored in sterile Whirl-Pak plastic bags, and kept at -20 C until processing.Total DNA isolation was performed with the FastDNA Soil Kit (MP Biomedicals). Briefly, 500 mg of soil samples were introduced into Lysing Matrix E tubes (containing 1.4 mm ceramic spheres, 0.1 mm silica spheres, and one 4 mm glass bead). Subsequently, mechanical lysis was performed using a FastPrep homogenizer (MP Biomedicals) with three pulses at 6 m/s for 45 s, placing the samples on ice between each pulse. The following steps were made according to the manufacturer's recommendations. The purified DNA was quantified by fluorometry using the Qubit dsDNA Broad Range (BR) kit (Invitrogen), and its integrity was corroborated by 1% agarose gel electrophoresis.Three different molecular markers were amplified and sequenced with the Illumina NovaSeq system using the services provided by Novogene. For bacterial analysis, we selected the V3-V4 region of the 16S rRNA gene (primers 341F 5'-CCTAYGGGRBGCASCAG-3' and 806R 5'-GGACTACNNGGGTATCTAAT-3'). For archaea, we targeted the V4-V5 region of the 16S rRNA gene (primers Arch519F 5'-CAGCCGCCGCGGTAA-3' and Arch915R 5'-GTGCTCCCCCGCCAATTCCT-3'). Additionally, for fungal analysis, we targeted the region ITS2 (primers ITS3-2024F 5'-GCATCGATGAAGAACGCAGC-3' and ITS4-2409R 5'-TCCTCCGCTTATTGATATGC-3').
SUB14820179
M4-B