GSM1478320: HP1_3; Homo sapiens; RNA-Seq
Blood specimens from 3 patients with PDAC were subjected to microfluidic depletion of RBCs and CD45 and CD66b-positive WBCs, leaving single CTCs and small CTC-clusters in the final product (pubmed ID 23552373). The product containing enriched cells was collected in a 35mm petri dish and viewed using a Nikon Eclipse Ti inverted fluorescent microscope. Cells of interest were identified based on intact cellular morphology and lack of labeling with anti-CD45 magnetic beads. These target cells were individually micromanipulated with a 10 μm transfer tip on an Eppendorf TransferMan NK 2 micromanipulator and ejected into PCR tubes containing RNA protective lysis buffer and immediately flash frozen in liquid nitrogen. 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. RNA-Seq using oligo-dT cDNA synthesis and amplification of cDNA libraries using custom universal PCR primers
GEO Accession
GSM1478320