title: GSM3685290: Mouse1_GFPPrimary_S48_clean; Mus musculus; OTHER
library_construction_protocol: Primary tumors were dissociated using collagenase D and trypsin. They were treated with ACK lysis buffer to remove contaminating red blood cells. These dissociated cells were stained with EpCam, CD45 antibodies. Single cells were isolated by flow cytometry for GFP+, EpCAM+, GFP+EpCAM+ and CD45+ from primary tumors. GFP+ cells were isolated from various metastases Single cells isolated into 10x Lysis Buffer with Rnase inhibitor. Next they were processed for mRNA transcription for first strand synthesis using Oligo-dT primer and cDNA generated is amplified for 15 cycles following SMART-Seq2 PCR primers. Samples were then treated ionically to fragment gDNA and cDNA to 400-700bp fragments. Later gDNA was amplified using SeqXE kit from Sigma Aldirch using manufacturer instructions. resulting cDNA and gDNA fragments are quantified.  Following this, libraries for sequencing were prepared by subjecting the 1ug of gDNA and cDNA fragments to end-repair, 3′ adenylation and ligation of TruSeq barcoded adapters. After ligation, DNA fragments were size selected using Ampure XP beads. A 0.75 ratio of beads to sample was used to select fragments larger than 200bp and avoid adapter dimers. These fragments were further amplified by PCR, cleaned using Qiaquick PCR Purification Kit and then once again size selected using Ampure XP beads with a 0.75 ratio of beads to sample. The final libraries were then quantified on Nanodrop and Bioanalyzer using Agilent's High Sensitivity DNA Analysis Kit. 10nM of each sample was then pooled together and the resulting pool was then sequenced single read 76bp length on the Illumina NextSeq
identifiers.namespace: GEO
identifiers.id: GSM3685290