title: GSM3711405: PLC 1; Homo sapiens; RNA-Seq
library_construction_protocol: The frozen cell pellet samples were dissolved in 6 ml Buffer RLT. Total RNA was isolated using the Rneasy Mini Kit (Qiagen) according to the manufacturer's instructions including on-column Dnase digestion. In order not to overload the silica column,  isolation was performed with 10 million cells (1/10 of the lysate=600 microliters). Total RNA was eluted in 30 microliters RNase-free water. Library preparation with the TruSeq Stranded mRNA HT technology, according to the manufacturer's protocol. The protocol started with an RNA fragmentation step using divalent cations. Samples were then introduced into a reverse transcription to generate first strand cDNA. In the second strand cDNA synthesis dUTP is incorporated instead of dTTP, to avoid amplification by the DNA polymerase in the subsequent PCR and thus guarantee strandedness. 3'-ends were adenylated and sequencing adapters were ligated. Those comprise sequencing primer- and flow cell-binding sites, as well as indices for multiplexed sequencing of pooled libraries. Adapter-ligated fragments were amplified during a limited-cycle PCR reaction.