Illumina HiSeq 4000 sequencing; RNA-seq analysis of ID8 ovarian cancer cells after harvest from malignant ascites
RNA-seq analysis of ID8 ovarian cancer cells after harvest from malignant ascites
Tumor cells from ascites was harvested by peritoneal lavage and collected by centrifugation. Subsequently tumor cells (ID8 cell line) were isolated by cell sorting. Single-cell suspensions were incubated at 4°C for 10 min with the 2.4G2 antibody for Fc receptor blocking followed by incubation with antibodies against CD45.2 (clone 104), CD11b (clone M1/70 details), F4/80 (clone BM8)CD64 (clone X54-5/7.1) CD169 (clone Ser4), Tim4 (clone RMT4-54), CD5 (clone 53-7.3), NK1.1 (clone PK136) and Ly6G (clone 1A8) for 30 min at 4°C. Prior to sort, cells were incubated with Sytox Blue (Thermo Fischer Scientific, FR) to discriminate dead cells and filtered through a 70 µm cell strainer. Tumor cells were then gated as Live (FSC-A vs Sytox Blue), CD45.2- (CD45.2 vs SSC-A), CD5neg, CD19neg (CD5 vs CD19), Linneg(NK1.1, Ly6G), CD11b- (CD11b vs Lineage), single cells (FSC-H vs FSC-W), CD64- and F4/80- (F4/80 vs CD64) and subsequently as large granulated cells (SSC-A vs FSC-A) Full length cDNA was generated using Clontech SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio Europe, Saint Germain en Laye, France) according to manufacturer's instructions from 500 cells isolated by fluorescence-activated cell sorting in PBS buffer containing RNAses inhibitor. cDNA was amplified with 14 cycles of PCR for cDNA amplification by Seq-Amp polymerase. Six hundred pg of pre-amplified cDNA were then used as input for Tn5 transposon tagmentation by the Nextera XT DNA Library Preparation Kit (96 samples) (Illumina, San Diego, CA) followed by 12 cycles of library amplification. Following purification with Agencourt AMPure XP beads (Beckman-Coulter, Villepinte, France), the size and concentration of library DNA were assessed on an Agilent 2100 Bioanalyzer.