title: GSM3822051: 5H_MV_rep2; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was isolated using the TriZol reagent method according to manufacturer's protocol. Libraries were prepared by UCL Genomics using the KAPA mRNA HyperPrep Kit (p/n KK8580) according to manufacturer's instructions Briefly, mRNA was isolated from total RNA using Oligo dT beads to pull down poly-adenylated transcripts. The purified mRNA was fragmented using chemical hydrolysis (heat and divalent metal cation) and primed with random hexamers. Strand-specific first strand cDNA was generated using Reverse Transcriptase in the presence of Actinomycin D. This allows for RNA dependent synthesis while preventing spurious DNA-dependent synthesis. The second cDNA strand was synthesised using dUTP in place of dTTP, to mark the second strand. The resultant cDNA is then "A-tailed" at the 3' end to prevent self-ligation and adapter dimerisation. Full length xGen adaptors (IDT), containing two unique 8bp sample specific indexes, a unique molecular identifier (N8) and a T overhang are ligated to the A-Tailed cDNA. Successfully ligated cDNA molecules were then enriched with limited cycle PCR (14 cycles – the actual number is dependent on the amount of input RNA) The high fidelity polymerase employed in the PCR is unable to extend through uracil. This means only the first strand cDNA is amplified for sequencing, making the library strand specific (first-strand)