GSM8112604: malignant tumor 3; Homo sapiens; miRNA-Seq
Total RNA was extracted using Absin's exosomal RNA extraction kit (abs60263).RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoDrop™ One/OneC (Thermo Fisher Scientific, CA, USA). RNA concentration was measured using Qubit® RNA HS Assay Kit in Qubit® 3.0 Flurometer (Life  Technologies CA, USA). RNA integrity was assessed using Agilent 4200 TapeStation (Agilent Technologies, CA, USA). Library preparation for small RNA sequencing A total amount of 3 µg RNA per sample was used as input material for the small RNA library preparations.  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, NEB 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_x0002_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 a 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 4200 TapeStation. Clustering and sequencing  The clustering of the index-coded samples was performed on a cBot Cluster Generation System using  TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster  generation, the library preparations were sequenced on an Illumina platform.