GSM2690850: Bulk tumor cells depleted of mouse - biological replicate 2; Homo sapiens; RNA-Seq
A total of 80,000 or more cells chosen for a specific surface marker status were collected and processed for RNA extraction. RNA was extracted using the Qiagen AllPrep Kit (Qiagen catalog #80204). Quality assessments were determined using Agilent BioAnalyzer and NanoDrop readings. Libraries were synthesized using the Illumina TruSeq Stranded mRNA sample preparation kit (Illumina # RS-122-2103). Total RNA samples were concentration normalized, and polyadenylated RNA was purified using oligo-dT attached to magnetic beads. Purified mRNA was fragmented using heat in the presence of divalent cations. Fragmented RNA was converted into double-stranded cDNA, with dUTP utilized in place of dTTP in the second strand master mix. A single ‘A’ base was added to the cDNA and forked adaptors that include index, or barcode, sequences were attached via ligation. The resulting molecules were amplified via polymerase chain reaction (PCR). Final libraries were quantified, normalized and pooled prior to binding to the flow cell. For multiplex sequencing, the following sample-Illumina barcode combinations were used: A2-D701, A3-D705, B1-D702, B2-D710, B3-706, C1-711, C2-703, C3-D707_507, D1-D712, D2-D704, D3-D708_507.