GSM1481228: smear under the skin graft (S) with Xen14 bacteria; Mus musculus; miRNA-Seq
Total RNA was extracted from the harvested serum using a mirVana miRNA Isolation Kit (Ambion, USA). The purified RNA yield was determined by the absorbance at 260 nm with an SSP-3000 NanoDrop spectrophotometer (Infinigen Biotech, USA), and RNA quality was evaluated with a BioAnalyzer 2100 system (Agilent Technologies, USA). RNA smaller than 200 base pairs (bp) was enriched with the mirVana miRNA isolation kit (Ambion). The small RNA samples were sent to GeneTech Biotech Co., Ltd (GeneTech, Taiwan) for small RNA cloning. The population of miRNAs with a length of 15–30 nucleotides (nt) was passively eluted from polyacrylamide gels. The RNA was then precipitated with ethanol and dissolved in water. Small RNAs had linkers ligated to them and bar-coded cDNAs were prepared using a TruSeq Small RNA Sample Prep Kit (Illumina, USA) following the manufacturer’s instructions. Individual libraries were analyzed on a BioAnalyzer (Agilent) for the presence of linked cDNA at the appropriate size (135–165 bp) and 11 bar-coded libraries were pooled into 1 sample by mixing 2.0 ng of the 135–165 bp peak from each sample, as determined by the BioAnalyzer. Sequencing of the pooled libraries was performed in 1 lane of the Illumina HiSeq2000 Sequencer. Fifty bp single-end reads of the libraries were obtained. After indexing and trimming of linker sequences, those reads of at least 15 nt in length that had less than three terminal mismatches in the sequence were sorted and counted for the following analysis. The clean read datasets were BLASTed with the BOWTIE software according to the following criteria: a 5′ and 3′ linker match of at least 15 nt and an appropriate length (15–29 nt). The pre-miRNAs and mature miRNAs in the miRBase v.20.0 were searched with BLAST to identify Mus musculus miRNAs.
GEO Accession
GSM1481228