title: GSM4419276: 30-P-sc; Mus musculus; RNA-Seq
library_construction_protocol: Single dormant (PKH26+) and proliferating (PKH26-) RM1536 from murine bone metastases were FACS-isolated following bioluminescence-imaging confirmation of tumor cells in bone at ~16 days post-intracardiac injection of PKH26-labelled RM1536 (eCFP+/luc2+) prostate cancer cells. Single tumor cells from bone marrow suspensions were sorted directly into a 384 well plate (containg 2 ml of RNA lysis bufer per well), sealed and snap-frozen on dry ice. Plates were stored at -80C until extraction. Single cell cDNA was generated using the SMARTer Ultra Low RNA Kit (Clontech) with modifications. Briefly, 1:2.5x106 dilution of ERCC spike-in controls (Ambion) were incorporated during first strand cDNA synthesis. Subsequent steps were performed according to manufacturer's instructions at half-reaction volumes. cDNA quality assessment using the Bioanalyzer HS DNA chip (Agilent technologies) according to manufacturer's instructions and via Qubit 4 (ThermoFisher). Libraries (1 ng cDNA input) were subsequently generated in four batches (2 x 14 samples of PKH+ cells; 2 x 16 samples of PKH- cells sourced from 8 independent animals at different time points of metastasis (average d16) across independent experiments) and unique adapters incorporated using the Nextera XT Kit (Illumina) according to manufacturer's protocol. Libraries were validated by Bioanalyzer 2100 (Agilent) and quantitated by Qubit 4 and pooled (2 x pools of equal numbers of PKH ± in each group) for sequencing across 2 lanes on the Illumina Hiseq2500.
attributes.tag: GEO Accession
attributes.value: GSM4419276