GSM2877959: Patient 1; Homo sapiens; RNA-Seq
For those samples with potential CTCs based on Imagestream analysis, the second aliquot was used for single-cell RNA sequencing (scRNAseq) using Chromium 10X technologies. The cell density was determined using a C-Chip DHC-N01 disposable hemocytometer. A mixture of 5 μl of 0.04% trypan blue (BioRad) and 5 μl of the single cell suspension was loaded unto the hemocytometer and examined at 10X magnification, using the Invitrogen Evos FL Cell Imaging System-digital inverted microscope. The single-cell chip loading, GEM generation & barcoding, post GEM-RT & cDNA amplification, and library construction were performed according to the ChromiumTM Single Cell 3' Protocol - Chemistry v2. For GEM generation an input of 10,000 cells total, at 1,000 cells/uL density, was targeted for each sample, with a target cell recovery of 6,000 cells. Library construction, enzymatic fragmentation, End-repair and A-tailing were performed as follows: pre-cool block at 4°C hold, fragmentation at 32°C for 5 minutes; End repair and A-tailing 65°C for 30 minutes and held at 4°C. Post reaction cleanup was performed, followed by Adaptor ligation. Adaptor ligation incubation was done at 20°C for 15 minutes. Post adaptor ligation cleanup was then performed, followed by sample index PCR with the following parameters: 98°C for 45 seconds; followed by 14 cycles: 98°C for 20 seconds; 54°C for 30 seconds; and 72°C for 20 seconds; followed by 72°C for 1 minute and held at 4°C. Quantification of the constructed libraries was evaluated using Qubit dsDNA HS Assay Kit (Thermo Fisher), Agilent cDNA High Sensitivity Kit, and Kapa DNA Quantification Kit for Illumina platforms, following the manufacturer's instructions. Generated libraries were sequenced on the Illumina HiSeq2500, using the paired-end 2 x 125bp sequencing protocol. Sequencing run parameters were set up according to version 2 chemistry, the number of cycles for each read as follows: Read 1: 26 cycles, i7 index: 8 cycles, i5 index: 0 cycles and Read 2: 98 cycles.