Residual DNA was removed by DNase I (Qiagen) Alpelisib solubility dmso digestion. We conducted a PCR with the digested RNA to exclude the possibility of residual DNA in downstream applications (PCR protocol see below). The concentration of extracted and purified RNA was determined spectrophotometrically
using a Nanodrop ND-1000 UV–vis spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). The integrity of the RNA was checked with an RNA 6000 picoassay on YM155 in vitro an Agilent 2100 Bioanalyzer (Agilent Technologies, Germany). To minimize extraction bias, total RNA from three individual filters (each representing 6-10 L water) per depth and sampling site were extracted. Total RNA was then reverse transcribed into cDNA using Qiagen’s QuantiTect Reverse Transcription kit and with random primers provided with the kit according to the manufacturer’s
instructions. After transcription of each individual sample, the three replicate transcribed products of each depth/sampling site were pooled and subjected to SSU cDNA amplification. First, amplification with EVP4593 nmr a ciliate specific primer set (Table 4) was performed to filter specifically the ciliate SSU rRNA from the env cDNA. The PCR reaction included 50–100 ng of template cDNA in a 50 μl-reaction, 1 U of Phusion High-Fidelity DNA polymerase (Finzymes), 1× Phusion HF Buffer, 200 μM of each deoxynucleotide triphosphate, and 0.5 μM of each oligonucleotide primer. The PCR protocol amplifying ca. 700 bp-long gene fragments consisted of an initial denaturation (30 s at 98°C) followed by 30 identical amplification cycles
(denaturation at 98°C for 10 s, annealing at 59°C for 10 s and extension at 72°C for 30 s), and a final extension at 72°C for 10 min. Subsequently, the purified (Qiagen’s MiniElute kit) PCR products from the first reaction were Florfenicol subjected to a second PCR, which employed eukaryote-specific primers for the amplification of the hypervariable V4 region (; Table 4). The PCR protocol started with 10 identical amplification cycles at an annealing temperature of 57°C where only the forward primer would operate, followed by 25 cycles with a primer annealing at 49°C where both forward and reverse V4 primers would amplify . The resulting PCR amplicons (ca. 480 bp) were excised from the gel using Qiagen’s Gel extraction kit. Gel extraction eliminates unspecific shorter fragments, invisible on a gel, in the final amplicon library. The integrity and length of purified amplicons was determined with a DNA 500 LabChip on an Agilent 2100 Bioanalyzer. Table 4 Primer sets used in this study for the specific amplification of ciliate V4-SSU rRNA fragments using a two-step (nested) PCR reaction Primer Primer sequences Reference 1.