Instrument: AB 5500xl Genetic Analyzer
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Blood specimens from 10 patients with metastatic breast cancer were subjected to microfluidic depletion of RBCs and CD45 and CD66b-positive WBCs, leaving single CTCs and small CTC-clusters in the final product (Ozkumur et al., 2013). Unfixed tumor cells were stained for cell surface expression of EpCAM, EGFR, HER2, MET, and the mesenchymal marker CDH11, all conjugated with an Alexa488 fluorophore, as well as stained with TexasRed-conjugated antibodies against CD45, CD14 and CD16 to identify contaminating leukocytes. Individual CTC-clusters (median of 3 cells per cluster) were isolated using a micromanipulator and compared with numerically matched pools of single CTCs from the same specimen, followed by next generation RNA sequencing (SOLiD 5500). Samples that showed high expression of contaminant leukocyte markers at the RNA level were excluded from the analysis. Libraries were constructed as previously described (Tang, F., Barbacioru, C., Nordman, E., Li, B., Xu, N., Bashkirov, V.I., Lao, K., and Surani, M.A. (2010). RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nat Protoc 5, 516-535). Briefly, to generate cDNA, samples were treated with reverse transcription master mix (0.05 uL RNase inhibitor, 0.07uL T4 gene 32 protein, and 0.33uL SuperScript III Reverse Transcriptase per 1X volume) and incubated on thermocycler at 50C for 30 minutes and 70C for 15 minutes. To remove free primer, 1.0uL of EXOSAP mix was added to each sample, which was incubated at 37C for 30 minutes and inactivated at 80C for 25 minutes. Next, a 3'-poly-A tail was added to the cDNA in each sample by incubating in master mix (0.6uL 10X PCR Buffer II, 0.36uL 25mM MgCl2, 0.18uL 100mM dATP, 0.3uL Terminal Transferase, 0.3uL RNase H, and 4.26uL H2O per 1X volume) at 37C for 15 minutes and inactivated at 70C for 10 minutes. A second strand cDNA was synthesis by dividing each sample into 4 and incubating in master mix (2.2uL 10X High Fidelity PCR Buffer, 1.76uL 2.5mM each dNTP, 0.066uL UP2 Primer at 100uM, 0.88uL 50mM MgSO4, 0.44uL Platinum Taq DNA Polymerase, and 13.654uL H2O per 1X volume) at 95C for 3 minutes, 50C for 2 minutes, and 72C for 10 minutes. DNA was sheared using a Covaris S2 system and then prepared for ABI 5500XL library construction with end polishing, size selection of 200-500 bp using AMPure XP, ABI barcode adaptor ligation, amplification and purification with AMPure XP, and then pooling of barcoded samples for emulsion PCR wiht template beads preparation. Samples were then loaded per protocol on the ABI 5500XL.