Fungal ITS gene library from pot experiment with clean sand and willows, growth time 30 days
Amplicon libraries for the for the fungi ITS2 (fITS2-C) were generated using up to 25 ng DNA as PCR template. PCR reaction (50L) contained 0.2mM dNTP mix, 0.01 units of Platinum SuperFi DNA Polymerase, and 500 nM primers containing custom 24nt barcodes followed by [ITS2-9] TACACACCGCCCGTCG and [ITS4] TCCTSCGCTTATTGATATGC, using a program of 98C 3min; 25cycles of 98C 30s, 62C 20s, 72C 2min; and 72C 7min, after which amplicons were purified with CleanNGS SPRI beads (3:5 ratio), eluted in 25 L nucleasefree water, quantified by Qubit, assessed by Tapestation 2200 and D1000/High sensitivity D1000 screentap for size and purity, and used to prepare sequencing libraries with the SQKLSK114 kit with 500 ng input and SPRIbased cleanups