Illumina TruSeq Stranded Total RNA Kit with Ribo-Zero Gold: Sample HCI019_pdx_P2_XXX_RNA_10962X22
Additional details of the library preparation process are described below: Library construction is performed using the Illumina TruSeq Stranded Total RNA Sample Preparation Kit with Ribo-Zero Human/Mouse/Rat (cat# RS-122-2201, RS-122-2202) as described below.  Briefly, Ribosomal RNA is removed from total RNA samples (100 ng to 4 ug) using biotinylated Ribo-Zero oligos attached to magnetic beads that are complimentary to cytoplasmic rRNA. Following purification, the rRNA-depleted sample is fragmented with divalent cations under elevated temperatures 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).  Follwoing 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.