LNCaP poly(A)+ RNA was extracted with FastTrack MAG Maxi mRNA Isolation Kit (Invitrogen), as per manufacturer’s protocol, treated with 25 U of DNase I, Amplification Grade (Invitrogen) for 1 h at room temperature, quantified using Quant-iT RiboGreen RNA Reagent (Invitrogen) and assessed for integrity on 2100 Bioanalyzer (Agilent). RNA samples from two biological replicates were processed and sequenced on Illumina HiSeq 2000.  Approximately 1 µg of this RNA was prepared for Next Generation Sequencing using a modified version of the Illumina protocol, Directional_mRNA-Seq_SamplePrep_Guide_15018460_A.pdf, in which 12 cycles of PCR were performed instead of 15 and standard Truseq adapters and Truseq barcoded primers were used instead of the v1.5 adapters and primers described in the guide.  A final size selection was performed using agarose gel to capture library with insert sizes ranging from 150 – 250 bases in length suitable for 2 x 75 base paired end sequencing. Library product was purified from agarose using standard oligo purification columns. The prepared library was then loaded onto an Illumina HiSeq v2 single-read flowcell, standard cluster generation was performed on a Cbot followed by paired-end sequenced of 2 x 75 bases of the insert and 7 bases of the index read using standard HiSeq sequencing reagents except for the use of an alternative read-2 sequencing primer which was designed as the reverse complement of the indexing read sequencing primer. Strand-oriented paired-end RNA-seq protocol.
GEO Accession
GSM2091092