GSM4100982: High dose [C1]; Homo sapiens; RNA-Seq
RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the Nano Photometer® spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using Qubit® RNA Assay Kit in Qubit®2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Approximately 3 μg RNA per sample was used as input material for the RNA sample preparations. First, ribosomal RNA was removed by using an Epicentre Ribo-zero rRNA Removal Kit (Epicentre, Madison, WI, USA), and rRNA-free residue was removed by ethanol precipitation. Subsequently, the high strand-specificity of the libraries was generated using the rRNA-depleted RNA of the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA), following manufacturer's recommendations. Briefly, fragmentation was conducted using divalent cations under elevated temperature in NEB Next. First-strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNaseH-). Second-strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP were replaced by dUTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3' ends of the DNA fragments, NEB Next adaptors with a hairpin loop structure were ligated to prepare for hybridization. To preferentially select cDNA fragments of 150–200 bp in length, the library fragments were purified with an AMPure XP system (Beckman Coulter, Brea, CA, USA). Then 3 μL USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then,PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers, and Index (X) Primers. Finally, the PCR products were purified (AMPure XP system), and library quality was assessed on an Agilent Bioanalyzer 2100 system. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA), following the manufacturer's instructions
GEO Accession
GSM4100982