title: GSM4823247: tumor tissue_HCC_2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA of each sample was extracted using TRIzol Reagent (Invitrogen)/RNeasy Mini Kit  (Qiagen).Total RNA of each sample was quantified and qualified by Agilent 2100 Bioanalyzer (Agilent   Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc.) and 1% agrose gel. 1μg   total RNA with RIN value above 7 was used for following library preparation. Next generation   sequencing library preparations were constructed according to the manufacturer's protocol (NEBNext®   Ultra™ Directional RNA Library Prep Kit for Illumina®). The rRNA was depleted from total RNA using Ribo-Zero™ rRNA removal Kit (Human/Mouse/Rat)  /(Yeast) /(Bacteria) (Illumina). The ribosomal depleted RNA was then fragmented and reverse-transcribed.   First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase with random primers and   Actinomycin D. The second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix   （include dACG-TP/dUTP）. The purified double-stranded cDNA by AxyPrep Mag PCR Clean-up   (Axygen) was then treated with End Prep Enzyme Mix to repair both ends and add a dA-tailing in one   reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA   was then performed using AxyPrep Mag PCR Clean-up (Axygen), and fragments of ~360 bp (with the   approximate insert size of 300 bp) were recovered. The dUTP-marked second strand was digested with  Uracil-Specific Excision Reagent (USER) enzyme (New England Biolabs). Each sample was then   amplified by PCR for 11 cycles using P5 and P7 primers, with both primers carrying sequences which can   anneal with flow cell to perform bridge PCR and P7 primer carrying a six-base index allowing for   multiplexing. The PCR products were cleaned up using AxyPrep Mag PCR Clean-up (Axygen), validated   using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by Qubit   2.0 Fluorometer (Invitrogen, Carlsbad, CA,USA). circular RNA-Seq; libraries with different indices were multiplexed and loaded on an Illumina HiSeq instrument   according to manufacturer's instructions (Illumina, San Diego, CA, USA). Sequencing was carried out  using a 2x150 paired-end (PE) configuration; image analysis and base calling were conducted by the   HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument.  The sequences were processed and analyzed by GENEWIZ.