GSM4706406: Her2(+)EpCAM(+) mammary tumor cells - CD4 and CD8-depleted  - Day20_rep4; Mus musculus; RNA-Seq
Tumors were collected at growth end-point (20 days post-injection of E-A14 tumor cells in CD4/CD8-depleted mice versus 47 days post-injection in control mice) and enzymatically digested in 10mg.mL-1 collagenase and 0.2mg.mL-1 DNAse I for 30min at 37°C. Red blood cells were then lysed 5min with Pharmlyse Buffer (BD Biosciences) at room temperature. These cells were labeled with fluorescence-conjugated CD90, CD105, CD45, Her2 and EpCAM antibodies and Zombie Fixable Viability Dye. Her2+EpCAM+ and Her2+EpCAM- cells were sorted by a FACSAria III cell sorter (BD Biosciences) after exclusion of dead cells, CD90(+), CD105(+) and CD45(+) cells. Total RNA was extracted from sorted cells and also from single cell suspensions of two replicates of in vitro cultured E-A14 tumor cells stabilized in RLT buffer according to the “Purification of total RNA from animal and human cells” protocol of the RNeasy Micro Kit (Qiagen). The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories) was used to generate first strand cDNA from 0.1 to 1.0 ng total-RNA. Double stranded cDNA was amplified by LD PCR (10, 12 or 14 cycles) and purified via magnetic bead clean-up. Library preparation was carried out as described in the Illumina Nextera XT Sample Preparation Guide (Illumina). The libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems). Equimolar amounts of each library were pooled, and the pools were used for cluster generation on the cBot with the Illumina TruSeq SR Cluster Kit v3. The sequencing run was performed on a HiSeq 1000 instrument using the indexed, 50 cycles single-read protocol and the TruSeq SBS v3 Reagents according to the Illumina HiSeq 1000 System User Guide.
GEO Accession
GSM4706406