Single-cell RNA sequencing (scRNA-Seq) allows the complete and unbiased analysis of the transcriptional state of an individual cell. In the past 5 years, scRNA-Seq contributed to the progress of the hematology field, advancing our knowledge of both normal and malignant hematopoiesis. Different scRNA-Seq methods are available, all relying on the conversion of RNA to cDNA, followed by amplification of cDNA in order to obtain a sufficient amount of genetic material for sequencing. Currently available scRNA-Seq platforms can be broadly divided into two categories droplet-based and plate-based. Each of these approaches has advantages and disadvantages that need to be considered when designing the experiment. Here, we describe detailed protocols of two of the most used methods for scRNA-Seq of hematopoietic cells Smart-Seq2 (plate-based) and 10× Genomics (droplet-based).Recurrent chromosomal translocations define genetic subtypes of childhood leukemia and present the first hit that generates an expanded clone of preleukemic cells in the bone marrow. Most commonly, reverse transcriptase PCR is used to detect these translocations on RNA level. This technique has severe drawbacks, including sensitivity to contamination and instability of RNA. Here, we describe the genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) that overcomes these pitfalls.Next-generation sequencing (NGS) of immunoglobulin (IG) and T cell receptor (TR) rearrangements represents a modern alternative to classical RQ-PCR-based minimal residual disease (MRD) detection. The same primer sets and conditions can be used for all patients, which is undoubtedly one of the most important benefits of NGS, not only reducing the labor required to perform the analysis but also enabling the assay to comply with the upcoming EU IVD regulation. So far, only one standardized academic protocol for this task has been published, developed, and validated within the EuroClonality-NGS working group. In this chapter we describe the materials and methods for amplicon library preparation for sequencing on Illumina MiSeq, and the bioinformatic pipeline for this protocol.Although new techniques (i.e., droplet digital-PCR, next-generation sequencing, advanced flow cytometry) are being developed, DNA-based allele-specific real-time quantitative (RQ)-PCR is still the gold standard for sensitive and accurate immunoglobulin/T cell receptor (IG/TR)-based minimal residual disease (MRD) monitoring, allowing the detection of up to 1 leukemic cell in 100,000 normal lymphoid cells. We herewith describe the standard PCR procedure which has been developed and standardized (with minor modification in single labs) through the last 20 years of activity of the EuroMRD Consortium, a volunteer activity of expert laboratories that is continuously providing education, standardization, quality control rounds, and guidelines for interpretation of RQ-PCR data.Mass cytometry is now a well-established method that enables the measurement of 40-50 markers (generally proteins but transcripts are also possible) in single cells. Analytes are detected via antibodies tagged with heavy metal and detected by using a time-of-flight mass spectrometer. Over the past decade, mass cytometry has proven to be a valuable method for immunophenotyping hematopoietic cells with remarkable precision in both healthy and malignant scenarios. This chapter explains in detail how to profile hematopoietic cells by using this high-dimensional multiplexed approach.Flow cytometry has been widely used in basic and clinical research for analysis of a variety of normal and malignant cells. Hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) can be highly purified by flow cytometry. Isolated HSCs and LSCs can be functionally identified by transplantation assays and can also be studied at the molecular level. Here we describe the flow cytometry methods for analysis and isolation of mouse HSCs and LSCs.The relative survival of cancer patients, when considering the tumoral stage at diagnosis, has not changed significantly in the last three decades, in spite of our increasingly detailed knowledge of the molecular alterations occurring in human tumors. In parallel, despite a growing number of clinical trials being conducted, the absolute number of drugs that are effective in humans is declining, and many new drugs move into the market without having enough evidence of their benefit on survival or quality of life. In part, this failure is due to the discordance between the results from preclinical and clinical trial phases, therefore leading to a high percentage of apparently promising lead compounds being abandoned in the transfer to the clinic.  https://www.selleckchem.com/products/Gefitinib.html  This discordance is caused, to a large degree, by the use of inappropriate animal models in the first stages of drug development. In this chapter, we discuss how the development of cancer therapies needs to be redesigned in order to achieve cancer cure, and how this redesign must involve the generation of better animal models, based on the tenets of the cancer stem cell theory, and capable of recapitulating all the aspects of human cancer. The use of such improved models should increase the likelihood of success in drug development, reducing the number of agents that go into trial, and the amount of patients undergoing useless trials.Only 10 years ago, the existence of cancer stem cells (CSCs) was still hotly debated. Even today, when their presence in most tumor types has been clearly demonstrated, all the consequences of their existence are far from being realized neither in the clinic nor, very often, in basic and translational cancer research. The existence of CSCs supposes a true change of paradigm in our understanding of cancer, but it will only have a real impact when we will properly assimilate its implications and apply these insights to both cancer research and cancer treatment. In this primer to the topic of leukemia stem cells (LSCs) our aim is to highlight with broad brushstrokes the most relevant of their properties, how these characteristics led to their identification, and the implications that the existence of LSCs has for the research and fight against leukemia.