GSM8085880: MM029 Lurbi Rep 1; Homo sapiens; RNA-Seq
P2683 The RNAs of all the samples have been extracted using the Macherey-Nagel NucleoSpin RNA Plus Kit (Ref. 740984.250) and stored at -80* P2683 : Total RNA-Seq libraries were generated from 700 ng of total RNA using TruSeq Stranded Total RNA Library Prep Gold kit and TruSeq RNA Single Indexes kits A and B (Illumina, San Diego, USA), according to manufacturer's instructions. Briefly, cytoplasmic and mitochondrial ribosomal RNA (rRNA) was removed using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. Following purification, the depleted RNA was fragmented into small pieces using divalent cations at 94oC for 8 minutes. Cleaved RNA fragments were then copied into first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity was achieved by replacing dTTP with dUTP during second strand synthesis. The double stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single 'A' nucleotide was added to the 3' ends of the blunt DNA fragments using a Klenow fragment (3' to 5'exo minus) enzyme. The cDNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR amplification (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 12 cycles; 5 min at 72oC). Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman-Coulter, Villepinte, France) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis.  P2988 : RNA-Seq libraries were generated from 500 ng of total RNA using TruSeq Stranded mRNA Library Prep Kit and IDT for Illumina - TruSeq RNA UD Indexes (Illumina, San Diego, USA), according to manufacturer's instructions with minor modifications. Following polyA selection, mRNAs were fragmented at 94oC for 8 minutes. DNA library were amplified using 12 cycles of PCR. Surplus PCR primers were further removed by purification using SPRIselect beads (Beckman-Coulter, Villepinte, France) and the final libraries were checked for quality and quantified using Bioanalyzer 2100 system (Agilent technologies, Les Ulis, France).  P3057 : Total RNA-Seq libraries were generated from 500 ng of total RNA. Before cDNA synthesis, cytoplasmic and mitochondrial ribosomal RNA (rRNA) were removed using a biotin-streptavidin magnetic bead-based procedure with the riboPOOL kit targeting h/m/r ribosomal rRNA (siTOOLs Biotech, Planegg/Martinsried, DE) according to manufacturer's instructions. Ribodepleted RNAs were purified using Agencourt RNAClean XP (Beckman-Coulter, Villepinte, France) and eluted in 8,5 µl ELB buffer (Illumina, San Diego, USA). Purified RNAs were then processed using Illumina TruSeq Stranded Total RNA Library Prep Gold and IDT for Illumina - TruSeq RNA UD Indexes, omitting the ribodepletion step and starting directly from the fragmentation step. RNAs were fragmented at 94oC for 8 minutes. DNA library were amplified using 13 cycles of PCR. Surplus PCR primers were further removed by purification using SPRIselect beads (Beckman-Coulter) and the final libraries were checked for quality and quantified using Bioanalyzer 2100 system (Agilent technologies, Les Ulis, France).