Small RNA Seq of Candida Parapsilosis + spike-ins
We used the TruSeq Small RNA Sample Preparation Kit (Illumina) to prepare the libraries for further sequencing. We initially performed a phosphatase treatment incubating 16 l of the digested samples for 30 min at 37 C and 5 min at 65 C mixed with 2.5 l of 10X phosphatase buffer, 2.5 l of nuclease-free water, 1 l of RNAse inhibitor and 3 l of Antarctic phosphatase (New England BioLabs Inc.). We then performed a kinase treatment adding 4 l of T4 Polynucleotide Kinase (PNK, New England BioLabs Inc.), 5 l of 10X PNK buffer, 10 l of ATP 10 mM, 1 l of RNAse inhibitor and nuclease-free water up to a total volume of 50 l, and incubating the samples 1 hour at 37 C. Samples were then purified using RNeasy MiniElute Cleanup kit following manufacturers instructions (Qiagen) with a 10 l RNase-free water final elution step. Samples were concentrated using a centrifugal evaporator Speed Vac to a final volume of 5 l and we started the TruSeq Small RNA Sample Preparation Kit (Illumina) protocol according to manufacturers instructions. After the final purification of the cDNA libraries, we performed quality control of each library using Agilent 2100 bioanalyzer with the DNA 1000 Kit (Agilent). Libraries were sequenced in single-reads with read lengths of 50 nucleotides in Illumina HiSeq2500 sequencers at the Genomics Unit of the CRG.
SUB9258707
17-ND-37_35281_GTAGAG