GSM8128913: AAV Liver, HBV Infected + Treatment, Day 38 rep 4; Mus musculus; RNA-Seq
polyA Illumina TruSeq Stranded mRNA sample preparation kit (Illumina # RS-122-2103) HiSeq-Sequencing-2x50bp-PE sequencing on an Illumina sequencing platform Briefly, Total RNA samples are concentration normalized, and polyadenylated RNA is purified using oligo-dT attached to magnetic beads. Purified mRNA is fragmented using heat in the presence of divalent cations. Fragmented RNA is converted into double-stranded cDNA, with dUTP utilized in place of dTTP in the second strand master mix. A single 'A' base is added to the cDNA and forked adaptors that include index, or barcode, sequences are attached via ligation. The resulting molecules are amplified via polymerase chain reaction (PCR). During PCR the polymerase stalls when a dUTP base is encountered in the template. Since only the second strand includes the dUTP base, this renders the first strand the only viable template, thereby preserving the strand information. Final libraries are quantified, normalized and pooled. Pooled libraries are bound to the surface of a flow cell and each bound template molecule is clonally amplified up to 1000-fold to create in- dividual clusters. Four fluorescently labeled nucleotides are then flowed over the surface of the flow cell and incorporated into each nucleic acid chain. Each nucleotide label acts as a terminator for polymerization, thereby en- suring that a single base is added to each nascent chain during each cycle. Fluorescence is measured for each cluster during each cycle to identify the base that was added to each cluster. The dye is then enzymatically removed to allow incorporation of the next nucleotide during the next cycle.