GSM9456077: FCM_1; Homo sapiens; RNA-Seq
Total RNA was extracted from the tissue using TRIzol® Reagent according to the manufacturer's instructions. RNA quality was assessed using a 5300 Bioanalyzer (Agilent) and quantified with an ND-2000 (NanoDrop Technologies). Only high-quality RNA samples [Concentration ≥ 20 ng/μL, total RNA > 1 μg, RQN > 4.5] were used for sequencing library construction. RNApurification, reverse transcription, library construction and sequencingwereperformed at Shanghai MajorbioBio-pharmBiotechnologyCo.,Ltd. (Shanghai,China)accordingtothemanufacturer'sinstructions.The XXRNA-seqtranscriptomelibrariywaspreparedfollowingIllumina®StrandedmRNAPrep,Ligation(SanDiego, CA) using1μgof totalRNA. Shortly,messengerRNAwas isolatedaccording topolyAselectionmethodby oligo(dT) beads and then fragmented by fragmentation buffer firstly. Secondly double-stranded cDNAwas synthesized with random hexamer primers. Then the synthesized cDNA was subjected to end-repair, phosphorylationandadapteradditionaccordingtolibraryconstructionprotocol.Librariesweresizeselectedfor cDNAtarget fragmentsof300-400bpusemagneticbeadsfollowedbyPCRamplifiedfor10-15PCRcycles.