概要: Purpose: Investigate cellular heterogeneity in a fresh human ovarian cancer tissue sample Methods: Enzymatic digestion of fresh tissue sample collected from the operating room to produce single cell suspension. Cells were labelled with fluorescent antibodies to CD3, CD14, CD19, CD20, CD56 and FACS sorted to remove immune cells. The negative population was used for sequencing. Single cells were processed using the Fluidigm C1 Chip to generate barcoded cDNA for each cell. Amplifed cDNA was sequenced using an Illumina HiSeq 2500 machine. Results: Single cell RNA sequence data was obtained for 92 cells and a "bulk" sample of 1000 cells. 26 cells were removed from analysis due to quality control standards. The remaining 66 cells and the bulk sample were analyzed. Conclusion: Single cell RNA sequence analysis reveals heterogeneity in gene expression in cells harvested from a high grade ovarian serous cancer
项目整体设计: A single cell suspension generated from a fresh high grade serous ovarian cancer sample was run through two Fluidigm C1 chips to isolate single cells and produce barcoded cDNA. Sequencing was performed in a single lane of an Illumina HiSeq 2500 machine. 92 single cells were sequenced and 1 bulk sample was sequenced, for a total of 93 samples.
Samples were dissociated using the Miltenyi Tumor Dissociation Kit and GentleMACs Dissociater (Miltenyi Biotec, Inc.). Red blood cells were lysed using Red Blood Cell Lysis buffer (Roche). Cells were fluorescently labelled using an antibody ccocktail containing antibodies to CD3, CD14, CD19, CD20 and CD56 (BioLegend, San Diego). Cells were flow sorted into negative (91%) and positive (9%) populations by flow cytometry. The negative population was subjected to single cell capture using the Fluidigm C1 chips following manufacturer's protocol
提取方案:
RNA extraction and barcoded cDNA generation was performed per Fluidigm protocol PN 100-7168 using Clontech cDNA and Nextera XT library reagents
建库方案:
-
测序信息
分子类型:
poly(A)+ RNA
库的片段类型:
PAIRED
库的链类型:
-
测序平台:
ILLUMINA
测序仪型号:
Illumina HiSeq 2500
链特异性:
Unspecific
样本
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疾病
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突变/变异
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Condition Detail
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文章
Single cell sequencing reveals heterogeneity within ovarian cancer epithelium and cancer associated stromal cells.
