title: GSM7913334: Blood_AD_Short Read 36; Homo sapiens; miRNA-Seq
library_construction_protocol: The blood was collected in PAXgene Blood RNA tubes. Total RNA was extracted following the protocol developed by this lab to isolate RNAs of different sizes and low concentration (Paz S, Mauer C, Ritchie A, Robishaw JD, Caputi M. A simplified SARS-CoV-2 detection protocol for research laboratories. PLoS One. 2020 Dec 18;15(12):e0244271). RNA sample quality and quantity were determined utilizing an Agilent TapeStation system and a NanoDrop spectrophotometer. The samples utilized for the cDNA library preparations had an RNA integrity number (RIN) ≥ 7 and an RNA concentration ≥ 30 ng/µL. Ribosomal RNA was removed by Epicentre TruSeq Stranded Total RNA Library Prep Gold/illumina/20020599 A total amount of 2 μg total RNA per sample was used as input material for the small RNA library. Sequencing libraries were generated using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA.) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, 3' SR Adaptor was directly and specifically ligated to 3' end of miRNA, siRNA and piRNA. After the 3' ligation reaction, the SR RT Primer hybridized to the excess of 3' SR Adaptor (that remained free after the 3' ligation reaction) and transformed the single-stranded DNA adaptor into a double-stranded DNA molecule. This step is important to prevent adaptor-dimer formation, besides, dsDNAs are not substrates for ligation mediated by T4 RNA Ligase 1 and therefore do not ligate to the 5´ SR Adaptor in the subsequent ligation step. 5´ends adapter was ligated to 5´ends of miRNAs, siRNA and piRNA. Then first strand cDNA was synthesized using M-MuLV Reverse Transcriptase (RNase H–). PCR amplification was performed using LongAmp Taq 2X Master Mix, SR Primer for Illumina and index (X) primer. PCR products were purified on an 8% polyacrylamide gel (100V, 80 min). DNA fragments corresponding to 140~160 bp (the length of small noncoding RNA plus the 3' and 5' adaptors) were recovered and dissolved in 8 μL elution buffer. At last, library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips.