Illumina TruSeq RNA  Library Preparation Kit v2 with poly(A) Selection: Sample HCI003_tumor_patient_XXX_RNA_8506X3
Intact poly(A) RNA was purified from total RNA samples (100-500 ng) with oligo(dT) magnetic beads and sequencing libraries were prepared as described using the Illumina TruSeq RNA Library Preparation Kit v2 (RS-122-2001, RS-122-2002).  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 10 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  RNA Library Preparation Kit v2 (RS-122-2001 and RS-122-2002) as described below,  Briefly, total RNA (100 ng to 500 ng) is poly-A selected using poly-T  oligo-attached magnetic beads.  Poly-A RNA is 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).  Following second strand synthesis, the cDNA is converted to blunt-ended fragments with 5'-phosphates and 3'-hydroxyl groups using a combination of enzymes that perform fill-in reactions and exhibit exonuclease activity.  An A-base is added to the blunt ends as a means to prepare the 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 cycles) and the amplified library is purified by Agencourt AMPure XP beads (Beckman Coulter Genomics cat#A63881).  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.