title: Bacterial_metagenome_block5_IceEncasement_Csampling
design: DNA was extracted from 100 mg of sieved, freeze-dried and homogenized soil samples with MoBio Power Soil DNA isolation kit. Bacterial amplicons were produced with tagged-519F and 926trP1 primers and fungal amplicons with tagged-ITS4 and fITS7 primers. Fungal and bacterial samples were amplified in triplicate 20 ul reactions. The PCR mixture contained 0.5 244  M of the primers, 0.2  M of each deoxynucleotide triphosphate, 0.4 U Phusion Hot Start 245 high fidelity II DNA polymerase (Thermo Fisher Scientific), 1 x Phusion GC Buffer and 20 ng of template DNA. PCR for bacterial amplification was initiated by incubation at 98  C for 3 min followed by 25 cycles: 10 s at 98  C, 30 s at 64  C and 30 s at 72  C, and a final extension for 5 min at 72  C. Amplification of fungal DNA was performed in two PRCR rounds. The first round was performed with ITS4 and fITS7 primer pair with the following PCR conditions: an initial denaturation at 98  C for 1 min followed by 20 cycles each consisting of 10 s at 98 C, 20 s at 54  C and 30 s at 72  C, and a final extension for 7 min at 72  C. One tenth of the first-round PCR product (2  l) was subjected to a second round of PCR comprising 15 cycles with conditions identical to those for first round except that the annealing temperature was 57  C and a tagged-ITS4 was used. PCR products were analyzed by MultiNA Microchip Electrophoresis System for DNA/RNA (Shimadzu). Triplicate amplicons were pooled in equimolar ratios and cleaned with AMPure XP  and quantified picogreen assay and diluted to a final concentration of 25 pM. Sequencing was performed at the Biocenter Oulu Sequencing Center with Ion Torrent PGM sequencer using Ion PGM OT2 400 bp template kit, Ion PGM Hi-Q Sequencing kit and 316 v2 chip.