title: GSM1150613: STT5668_Breast_020_normal; Homo sapiens; RNA-Seq
library_construction_protocol: Multiple 2 mm-diameter core punches were taken from FFPE tissue blocks to collect dense areas of neoplasia and adjacent normal breast epithelial content separately.  When ductal carcinoma in situ (DCIS) and/or invasive ductal carcinoma (IDC) were present, these were also cored.  Three to ten tissue cores were pooled for each diagnosis per patient, and tissue cores were re-embedded in paraffin and re-evaluated histologically for lesion purity by sectioning length-wise along the core.  Only samples which possessed >90% of luminal cells with the appropriate diagnosis within the epithelial compartment were included among our samples.  Adjacent normal cores contained no identifiable pathologies, and cancer cores possessed less than 5% early neoplasia or normal epithelial cells.  In all cases stromal cells were present in the core samples.  DNA and total RNA were purified from paraffin slices of the embedded tissue cores following deparaffination with a xylene incubation, ethanol wash, and a protease digestion, using the RecoverAll Total Nucleic Acid Isolation Kit (Ambion/Life Technologies; catalog #1975). 3SEQ libraries were prepared from as little as 5 ug total RNA.  RNA was enriched for 3’ ends by using the Oligotex mRNA mini Kit (QIAgen).  The 3’- poly(A)-enriched RNA pools were made into directional 3SEQ Illumina sequencing libraries as follows.  RNA that was not sufficiently fragmented was heat-sheared to a size of approximately 100-200 bases by incubation with First Strand Buffer (Invitrogen/Life Technologies) and oligo-dT-P7 primer (5’- CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC TTT TTT TTT TTT TTT TTT TTT TTT TVN -3’) at 85oC for 3-5 minutes, depending on the extent of fragmentation required.  The mixture was cooled to 50oC and first strand cDNA synthesis was performed in a 20 ul reaction using Superscript III Reverse Transcriptase (Invitrogen/Life Technologies) for 1 hour at 50oC.  Second strand cDNA was synthesized using Second Strand Buffer (Invitrogen/Life Technologies), DNA ligase (Invitrogen/Life Technologies), DNA polymerase I (New England Biolabs), and RNaseH (Epicentre Biotechnologies/Illumina), and purified using the QIAquick PCR Purification Kit (QIAgen).  3’ end repair and modification used dATP, Klenow exo (New England Biolabs) and Klenow Buffer, and was purified using the QIAgen MinElute PCR Purification Kit (QIAgen).  cDNA was then ligated to the double-stranded P5 Linker (Adapter Oligo Mix; Illumina) for 15 minutes at 22oC.  SPRI bead purification (Agencourt Biosciences/Beckman Coulter) was performed both prior to and following PCR amplification of the linker-ligated cDNA using 5' - AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T - 3' and 5' - CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC - 3' primers and the Phusion PCR Master Mix (New England Biolabs) using a 15 cycle program (98oC for 30 sec; 15 cycles of 98oC for 10 sec, 65oC for 30 sec, 72oC for 30 sec; 72oC for 5 min).  The libraries were then size selected for 200–300 bp fragments by agarose gel fractionation (3% Nusieve GTG; Lonza/Fisher Scientific) and purified using the QIAquick Gel Extraction Kit (QIAgen).  The libraries contain P7 sequence at the 3’ end downstream of poly(A) and P5 at the 5’ end.