Illumina TruSeq Stranded mRNA Library Preparation Kit with poly(A) selection: Sample HCI024_pdx_PX_XXX_RNA_14308X3
Intact poly(A) RNA was purified from total RNA samples (100-500 ng) with oligo(dT) magnetic beads and stranded mRNA sequencing libraries were prepared as described using the Illumina TruSeq Stranded mRNA Library Preparation Kit (RS-122-2101, RS-122-2102).  Purified libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay (cat# 5067-5582 and 5067-5583).  The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Biosystems Kapa Library Quant Kit (cat#KK4824).  Individual libraries were normalized to 5 nM and equal volumes were pooled in preparation for Illumina sequence analysis.      Additional details of the library preparation process are described below: Library construction is performed using the Illumina TruSeq Stranded mRNA Library Preparation Kit (cat# RS-122-2101, RS-122-2102) as described below.  Briefly, total RNA (100 ng to 4 ug) is poly-A selected using poly-T  oligo-attached magnetic beads.  Poly-A RNA eluted from the magnetic beads is fragmented and primed with random hexamers in preparation for cDNA synthesis.  First strand reverse transcription is accomplished using Superscript II Reverse Transcriptase (Invitrogen cat#18064-014).  Second strand cDNA synthesis is accomplished using DNA polymerase I and Rnase H under conditions in which dUTP is substituted for dTTP, yielding blunt-ended cDNA fragments in which the second strand with dUTP.  An A-base is added to the blunt ends as a means to prepare the cDNA fragments for adapter ligation and block concatamer formation during the ligation step.  Adapters containing a T-base overhang  are ligated to the A-tailed DNA fragments.  Ligated fragments are PCR-amplified (12-15 cycles) under conditions in which the pcr reaction enables amplification of the first strand cDNA product, whereas attempted amplifciation of the second strand product stalls at dUTP bases and therefore is not represented in the amplified library.  The PCR-amplified library is purified using Agencourt AMPure XP beads (Beckman Coulter Genomics cat#A63881).  Following amplification, the library is purified by bead based methodologies.  The concentration of the amplified library is measured with a NanoDrop spectrophotometer and an aliquot of the library is resolved on an Agilent 2200 Tape Station using a D1K (cat# 5067-5361 and 5067-5362) or a High Sensitivity D1K (cat# 5067-5363 and 5067-5364) assay to define the size distribution of the sequencing library.  Libraries are adjusted to a concentration of approximately 10 nM and quantitative PCR is performed using the KapaBiosystems Kapa Library Quant Kit (cat# KK4824) to calculate the molarity of adapter ligated library molecules.  The concentration is further adjusted following qPCR to prepare the library for Illumina sequence analysis.