概要: By transcriptionally profiling single cells from patients spanning different stages of lung adenocarcinoma progression, we provide evidence of epithelial regeneration in untreated primary tumours and a continuum of phenotypic states spanning key stages of embryonic development and lung morphogenesis in metastases. We transcriptionally profiled 41,384 single cells obtained from fresh surgical human samples taken from non-tumour-involved lung (n = 4), primary lung adenocarcinomas (n = 8; 7 untreated and 1 post neo-adjuvant chemotherapy), as well as brain (n = 3), bone (n = 1), and adrenal (n = 1) lung adenocarcinoma metastases. These samples were derived from patients spanning various stages of tumour progression without enrichment for a specific cell type, such that the entire tumour and its microenvironment were sampled in an unbiased manner. Data from all patients were merged to create a global cell atlas of the normal lung, primary tumours, and metastases.
项目整体设计: Single-cell RNA sequencing was performed on 17 donors using the 10X Genomics protocol. For each donor, all viable cells (identified by forward and side scatter and DAPI exclusion) were assayed for trancriptome-wide RNA-sequence.
Non-involved lung, tumour tissues, and metastatic lesions were obtained from patients with lung adenocarcinoma undergoing resection surgery at Memorial Sloan Kettering Cancer Center (MSKCC, New York, NY) after obtaining informed consent. Tissue samples were immediately placed in RPMI media (Corning) or Hypothermosol on ice and dissociated using both mechanical and enzymatic digestion (Human Tumour Dissociation Kit #130-095-929, Miltenyi Biotec), generally within 1 h of surgical resection. Tissues were minced with a razor blade in the Miltenyi enzyme mix according to the manufacturer's specifications and transferred to a Gentle MACS Octo Dissociator with heaters (# 30-096-427, 37℃) for further mechanical dissociation. Upon completion, cell suspensions were passed through a 70 μm filter and washed twice with FACS buffer (2% heat-inactivated FBS, 1 mM EDTA and Pen/Strep in PBS without Ca or Mg). Red blood cells were lysed in Red Blood Cell Lysis Solution (ACK lysis buffer) once or twice depending on red blood cell content, and final single-cell suspensions were made in Hanks’ Balanced Salt Solution (HBSS). For scRNA-seq, the remaining cell suspensions were subsequently flow sorted with a BD FACSAria II cell sorter fitted with a 100 μM nozzle to enrich for viable, single cells according to forward and side scattering, and DAPI exclusion. Cells were sorted directly into RPMI media with 10% FBS, washed thrice and re-suspend in PBS with 0.04% BSA for single cell encapsulation.
建库方案:
The 10X Genomics Chromium Platform was used to generate a targeted 5000 single cell Gel Bead-In-Emulsions(GEMs) per sample, loaded with an initial cell viability of ~90%. scRNA-seq libraries were prepared following the 10X Genomics user guide (Single Cell 3’Reagent Kits User Guide PN-120233, 10X Genomics).
测序信息
分子类型:
poly(A)+ RNA
库的片段类型:
PAIRED
库的链类型:
Forward
测序平台:
ILLUMINA
测序仪型号:
Illumina HiSeq 2500
链特异性:
Specific
样本
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数据来源
GEN样本编号
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疾病
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Condition Detail
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加标（Spike-In）
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平均测序片段长度_1
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文章
Regenerative lineages and immune-mediated pruning in lung cancer metastasis.
