概要: To understand molecular events associated with myeloma progression, we applied single-cell RNA sequencing to myeloma cells in the bone marrow and extramedullary sites. Transcriptome analysis, at single cell level, demonstrates that proliferation and alterations in antigen presentation are associated with myeloma progression whereas NF-kB pathway activation has an anti-correlation. While these alterations are prominent in most of the extramedullary myeloma cells, some myeloma cells in the bone marrow show gene expression signatures resembling extramedullary cells even when the myeloma cells were taken at stable disease. Altogether single-cell transcriptome analysis reveals both common transcriptional programs and heterogeneity turned on during myeloma progression.
项目整体设计: We performed single cell RNA sequencing (RNA-seq) for multiple myeloma from the bone marrow and/or extramedullary sites from 10 patients. Additional bulk RNA sequencing was performed with CD138-negative non-myeloma populations from the same patients. Data contain 477 single-cell RNA-seq and 11 bulk RNA-seq.
Plasma cell were purified using EasySep(®) CD138 Positive Selection kit before loading to integrated fluidic circuit (IFC) chip. Each suspension was loaded to 5-10um IFC for mRNA sequencing chip then cDNA synthesis and amplification were performed with SMARTer Ultra Low RNA Kit (Clontech) in the C1TM Single-Cell Auto Prep System (Fluidigm). Two types of external sequence, 1,4,7 ArrayControl TM RNA Spikes and ERCC RNA Spike-In Control Mix (ThermoFisher) were added to the lysis mix for validation of array and batch effect. All of amplified cDNAs were quantified and qualified by the Qubit® 2.0 Fluorometer (Life Technologies) and 2100 Bioanalyzer (Agilent Technologies).; Plasma cells were purified using EasySep(®) CD138 Positive Selection kit before loading to integrated fluidic circuit (IFC) chip. Each suspension was loaded to 5-10um IFC for mRNA sequencing chip then cDNA synthesis and amplification were performed with SMARTer Ultra Low RNA Kit (Clontech) in the C1TM Single-Cell Auto Prep System (Fluidigm). Two types of external sequence, 1,4,7 ArrayControl TM RNA Spikes and ERCC RNA Spike-In Control Mix (ThermoFisher) were added to the lysis mix for validation of array and batch effect. All of amplified cDNAs were quantified and qualified by the Qubit® 2.0 Fluorometer (Life Technologies) and 2100 Bioanalyzer (Agilent Technologies).
建库方案:
Whole-transcriptome sequencing libraries were generated for the RNAs from CD138-negative cell fractions using a TruSeq RNA Sample Preparation v2 Kit (Illumina).; Sequencing libraries were constructed using amplified cDNAs with the Nextera XT DNA Sample Prep Kit (Illumina) and sequenced using the 100 bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS Kit (Illumina).
测序信息
分子类型:
poly(A)+ RNA; rRNA- 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; Unspecific
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文章
Alterations in the Transcriptional Programs of Myeloma Cells and the Microenvironment during Extramedullary Progression Affect Proliferation and Immune Evasion.
Clinical cancer research : an official journal of the American Association for Cancer Research . 2019-09-26 [PMID:
31558476]
