Leukemia Stem Cells: Methods and Protocols

Preface
Contents
Contributors
Part I: Introductory Reviews
	Chapter 1: Introduction and Classification of Leukemias
		1 Introduction
		2 Classification of Leukemias
			2.1 Stratification Based on Histology and Surface Phenotype
			2.2 Stratification Based on Cytogenetics and Molecular Analyses
			2.3 Next-Generation Stratification
		3 Re-writing our Understanding of Hematopoiesis
		4 The Principle of Leukemia
		5 The Nature of Leukemia Cells
		6 Concluding Remarks
		References
	Chapter 2: Leukemia Stem Cells: Concept and Implications
		1 Introduction
		2 The Unmet Need of Cancer Cures
		3 Tumors Are Heterogeneous Tissues: The Road to Leukemia Stem Cell Discovery
		4 The Leukemia-Initiating Cell and the Preleukemic Stem  Cell
		5 Implications of the Existence of LSCs
			5.1 Implications for Leukemia Treatment
			5.2 Implications for Leukemia Research
				5.2.1 Implications for Research with Human Samples
				5.2.2 Implications for Leukemia Modeling in Animals
		6 Conclusions
		References
	Chapter 3: Leukemia Stem Cell Drug Discovery
		1 Introduction
		2 From Conventional Chemotherapy to Targeted Approaches
		3 Cancer Stem Cells: The Hidden Hand Behind Cancer
		4 CSC-Based Animal Models of Cancer: Essential Tools in Translational Medicine
		5 Current Mouse Models of Human Cancer
		6 How to Model Human Cancer in Mice?
		7 Conclusions
		References
Part II: Protocols
	Chapter 4: Analysis and Isolation of Mouse Leukemic Stem Cells
		1 Introduction
		2 Materials
		3 Methods
			3.1 Isolation of  HSCs
				3.1.1 Collecting BM Cells
				3.1.2 Enrichment by  MACS
				3.1.3 Antibody Staining
				3.1.4 Sorting of  HSCs
			3.2 Methods for Isolation of LSCs from  Mice
				3.2.1 Isolate LSCs from MLL-AF9-Induced AML  Mice
				3.2.2 Isolate LSCs from ICN-1-Induced T-ALL  Mice
				3.2.3 Isolate LSCs from N-Myc-Induced B-ALL  Mice
		4 Notes
		References
	Chapter 5: Mass Cytometry of Hematopoietic Cells
		1 Introduction
		2 Materials
			2.1 Thawing and Viability Treatment of Cells
			2.2 Stimulation and Fixation of Cells
			2.3 Barcoding of Fixed Cells
			2.4 Staining Fixed Cells
			2.5 CyTOF Acquisition
		3 Methods
			3.1 Thawing and Viability Treatment of Cryopreserved Bone Marrow Mononuclear Cells
			3.2 Stimulation and Fixation of Bone Marrow Mononuclear Cells
			3.3 Barcoding of Fixed Cells
			3.4 Staining Fixed Cells
			3.5 CyTOF Acquisition
		4 Notes
		References
	Chapter 6: PCR Technology to Identify Minimal Residual Disease
		1 Introduction
		2 Materials
		3 Methods
			3.1 PCR Screening of IG/TR Rearrangements
				3.1.1 PCR Mixes
				3.1.2 PCR Mix Composition for IGH, IGK, TRD, and TRG (see Note 2)
				3.1.3 PCR Mix Composition for  TRA
				3.1.4 PCR Mix Composition for  TRB
				3.1.5 PCR Set  up
				3.1.6 PCR Amplification Conditions and Verification of PCR Amplification
			3.2 Assessment of Clonality and Sequencing of Clonal IG/TR Rearrangements
				3.2.1 Homo-Heteroduplex Gel Electrophoresis (See Also Note 3)
				3.2.2 Sequencing and Identification of Clonal Patient-Specific Variable Regions
			3.3 Sequence Interpretation and Design of Allele-Specific Oligonucleotides for RQ-PCR
			3.4 RQ-PCR Sensitivity Testing and MRD Monitoring
			3.5 MRD Monitoring by RQ-PCR
				3.5.1 DNA Quantification Using a Reference Gene (Albumin)
				3.5.2 MRD Quantification in Follow-up Samples
				3.5.3 Interpretation of MRD Results
			3.6 Reporting MRD Values to Clinicians
		4 Notes
		References
	Chapter 7: Next-Generation Sequencing Technology to Identify Minimal Residual Disease in Lymphoid Malignancies
		1 Introduction
		2 Materials
			2.1 1st  PCR
			2.2 Purification of TRB-VJ and TRB-DJ PCR Products by Gel Extraction
			2.3 2nd  PCR
			2.4 Purification of Subpools by Gel Extraction
			2.5 Preparation of the Final Pool for Sequencing, and Sequencing
		3 Methods
			3.1 1st  PCR
			3.2 Purification of TRB-VJ and TRB-DJ PCR Products by Gel Extraction
			3.3 2nd  PCR
			3.4 Purification of Subpools by Gel Extraction
			3.5 Preparation of the Final Pool for Sequencing, and Sequencing
			3.6 Bioinformatic Analysis of the  Data
		4 Notes
		References
	Chapter 8: Genomic Inverse PCR for Screening of Preleukemic Cells in Newborns (GIPFEL Technology)
		1 Introduction
		2 Materials
			2.1 CD19+ Enrichment
			2.2 DNA Isolation
			2.3 Restriction Enzyme Digest
			2.4 Ligation
			2.5 Exonuclease Digest
			2.6 Ethanol Precipitation
			2.7 PCR/qPCR Reactions
			2.8 Gel Electrophoresis
			2.9 Gel Extraction
			2.10 Sanger Sequencing
		3 Methods
			3.1 CD19+ Enrichment
			3.2 DNA Isolation
			3.3 Restriction Enzyme Digest
			3.4 Cleanup of Restriction Enzyme Digest
			3.5 Ligation
			3.6 Exonuclease Digest
			3.7 Ethanol Precipitation
			3.8 Pre-amplification PCR
			3.9 Semi-Nested Real-Time  PCR
			3.10 Demultiplexed Semi-Nested Real-Time PCR (See Note 19)
			3.11 Demultiplexed Semi-Nested  PCR
			3.12 Agarose Gel-Electrophoresis
			3.13 Gel Extraction
			3.14 Sanger Sequencing
		4 Notes
		References
	Chapter 9: Single-Cell Transcriptomic Analysis of Hematopoietic Cells
		1 Introduction
		2 Materials
			2.1 Tissue Dissociation
			2.2 SmartSeq2
				2.2.1 Cell Lysis
				2.2.2 Reverse Transcription
				2.2.3 Amplification
				2.2.4 Cleaning and Quality Control
				2.2.5 Library Preparation
				2.2.6 Library Cleaning and Quality Control
			2.3 10x Genomics
				2.3.1 Gel Bead-in-EMulsions (GEM) Generation and Barcoding (See Note 4)
				2.3.2 Post GEM-RT Cleanup and cDNA Amplification
				2.3.3 3′ Gene Expression Library Construction
			2.4 Library Quantification, Pooling, and Sequencing
		3 Methods
			3.1 Tissue Dissociation
			3.2 SmartSeq2
				3.2.1 Cell Lysis
				3.2.2 Reverse Transcription
				3.2.3 Amplification
				3.2.4 Cleaning and Quality Control
				3.2.5 Library Preparation
				3.2.6 Library Cleaning and Quality Control
			3.3 10x Genomics
				3.3.1 GEM Generation and Barcoding
				3.3.2 Post GEM-RT Cleanup and cDNA Amplification
				3.3.3 3′ Gene Expression Library Construction
			3.4 Library Quantification, Pooling, and Sequencing
				3.4.1 Library Quantification
				3.4.2 Sequencing
		4 Notes
		References
	Chapter 10: In-Depth Mass Spectrometry-Based Single-Cell and Nanoscale Proteomics
		1 Introduction
		2 Materials
			2.1 Chip Fabrication
			2.2 Cell Culture Solutions
			2.3 Single-Cell Selection and Cell Lysis
			2.4 Cell Digestion and Sample Collection
			2.5 Liquid Chromatography
				2.5.1 Preparation of Analytical, Split Flow, and Solid-Phase-Extraction (SPE) Columns
				2.5.2 Etching and Connecting Emitter  Tip
				2.5.3 Liquid Chromatography-Mass Spectrometry Setup
				2.5.4 Other Accessories
				2.5.5 Buffers
		3 Methods
			3.1 Fabrication of Nanowell  Chip
			3.2 Fabrication of Cover Plate
			3.3 Cell Culture and Harvesting
			3.4 Single-Cell Selection and Cell Lysis Using nanoPOTS
			3.5 Cell Digestion and Sample Collection
			3.6 Packing of Analytical, Split, and SPE Columns
				3.6.1 Fabricate Frit Using the Frit  Kit
				3.6.2 Packing the Analytical and Split Columns
				3.6.3 Packing the  SPE
			3.7 Preparing a Chemically Etched Fused Silica Capillary nanoESI Emitter (See Note 1)
			3.8 LC-MS Setup
		4 Notes
		References
	Chapter 11: In Vivo Clonal Analysis of Aged Hematopoietic Stem Cells: Single-Cell Transplantation
		1 Introduction
		2 Materials
			2.1 Isolation of Bone Marrow Cells
			2.2 Staining HSCs in the Bone Marrow (See Note 2)
			2.3 Single-Cell Sorting of  HSCs
			2.4 Single-Cell Transplantation of Sorted  HSCs
			2.5 Peripheral Blood Analysis
		3 Methods
			3.1 Isolation of Bone Marrow Cells
			3.2 Staining HSCs in the Bone Marrow
			3.3 Single-Cell Sorting of  HSCs
			3.4 Single-Cell Transplantation of Sorted  HSCs
			3.5 Peripheral Blood Analysis
		4 Notes
		References
	Chapter 12: Experimental Competitive Bone Marrow Transplant Assays
		1 Introduction
		2 Materials
			2.1 Preparation of Recipient Mice
			2.2 Preparation of BM Cells
			2.3 Flow Cytometry for HSC Equivalents
			2.4 Preparation of Bone Marrow Cells for Transplant
			2.5 Bone Marrow Transplants
			2.6 Peripheral Blood Analysis
			2.7 Analysis of BM Reconstitution
		3 Methods
			3.1 Preparation of Recipient Mice
			3.2 Preparation of Donor and Competitor Bone Marrow (BM) Cell Samples
			3.3 Flow Cytometry Staining to Establish Donor Cells HSC Equivalents
			3.4 Preparation of the Bone Marrow Cells for Transplant
			3.5 Bone Marrow Transplants
			3.6 Peripheral Blood Analysis
			3.7 Analysis of BM Reconstitution
			3.8 Secondary and Tertiary Grafts
		4 Notes
		References
	Chapter 13: Human T-ALL Xenografts
		1 Introduction
		2 Materials
			2.1 Leukemic Blasts Enrichment by Density Centrifugation
			2.2 Phenotypic Characterization by Flow Cytometry
			2.3 Cryopreservation
			2.4 Lentiviral Transduction
			2.5 Mouse Immunodeficient Strains and Irradiation
			2.6 Cell Inoculation
				2.6.1 Retro-Orbital Injection
				2.6.2 Intravenous Injection
				2.6.3 Subcutaneous Injection
			2.7 Therapeutic Treatment of T-ALL Xenotransplanted Mice
				2.7.1 Intraperitoneal Administration
				2.7.2 Oral Gavage Administration
			2.8 Xenograft Monitoring and Mouse Analysis
				2.8.1 Body Weight Measurement
				2.8.2 Peripheral Blood Extraction
				2.8.3 Bone Marrow Aspiration
				2.8.4 Bioluminescence Imaging (IVIS)
				2.8.5 Subcutaneous Tumor Measurement and Analysis
				2.8.6 Mice Euthanasia and Organ Extraction
		3 Methods
			3.1 T-ALL Sample Preparation, Characterization, and Lentiviral Transduction
				3.1.1 Enrichment of Leukemic Blasts from Peripheral Blood or Bone Marrow Samples by Density Centrifugation
				3.1.2 Phenotypic Characterization by Flow Cytometry
				3.1.3 Cryopreservation
				3.1.4 Lentiviral Transduction
			3.2 T-ALL Xenotransplantation
				3.2.1 Mouse Immunodeficient Strains and Irradiation
				3.2.2 Cell Inoculation
			3.3 Therapeutic Treatment of T-ALL Xenotransplanted Mice
				3.3.1 Intraperitoneal Administration
				3.3.2 Oral Gavage Administration
			3.4 Xenograft Monitoring and Mouse Analysis
				3.4.1 Body Weight Measurement
				3.4.2 Peripheral Blood Extraction
				3.4.3 Bone Marrow Aspiration
				3.4.4 Bioluminescence Imaging (IVIS)
				3.4.5 Subcutaneous Tumor Measurement and Analysis
				3.4.6 Mice Euthanasia and Organ Extraction for Flow Cytometry Analysis
		4 Notes
		References
	Chapter 14: An Experimental and Computational Protocol to Study Cell Proliferation in Human Acute Myeloid Leukemia Xenografts
		1 Introduction
		2 Materials
			2.1 Generation of AML Patient-Derived Xenografts
			2.2 In Vivo Label-Retaining Assay
			2.3 Flow Cytometry: Data Acquisition and Analysis
			2.4 Computational Analysis of Cell Proliferation with ProCell
			Box 1 ProCell Installation
		3 Methods
			3.1 Generation of AML Patient-Derived Xenografts (Fig. 1, Steps 1 and 2)
			3.2 In Vivo Label-Retaining Assay (Fig. 1, Steps 3-5)
			3.3 Flow Cytometry: Data Acquisition and Analysis (Fig. 1, Steps 6 and 7)
			3.4 Computational Analysis of Cell Proliferation with ProCell (Fig. 1, Steps 8 and 9)
		4 Notes
		References
	Chapter 15: Leukemic Stem Cell Culture in Cytokine-Free Medium
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Flow Cytometry
		3 Methods
			3.1 Primary Human AML Cell Culture
			3.2 Flow Cytometry
		4 Notes
		References
	Chapter 16: Ex Vivo Expansion of Adult Hematopoietic Stem and Progenitor Cells with Valproic Acid
		1 Introduction
		2 Materials
			2.1 Isolation of CD34+ Cells from  UCBs
			2.2 Cytokines, Antibodies, and  Kits
			2.3 Cryopreservation
		3 Methods
			3.1 Density Gradient Isolation of Mononuclear Cells
			3.2 CD34+ Cell Purification from UCBs (See Note 13)
			3.3 Ex Vivo Expansion of Purified UCB-CD34+ Cells with  VPA
			3.4 Antibody Staining for Flow Cytometry Analysis
			3.5 Flow Cytometry Acquisition and Data Analysis
			3.6 Cryopreservation of Purified UCB-CD34+ Cells or Ex Vivo-Expanded Cells with  VPA
		4 Notes
		References
	Chapter 17: Isolation, Culture, and Manipulation of Human Cord Blood Progenitors
		1 Introduction
		2 Materials
			2.1 Human Mononuclear Cells Isolation from Cord Blood
			2.2 CD34+ HSPCs Cell Isolation
			2.3 CD34+ HSPCs Cells Cryopreservation
			2.4 CD34+ HSPCs Culture
			2.5 CD34+ HSPCs Transduction and Sorting
			2.6 Colony-Forming Unit Assay
			2.7 Cell Proliferation Assay
			2.8 Mice Transplantations and Follow-Up
			2.9 Analysis of Human Engraftment in  Mice
		3 Methods
			3.1 Cord Blood Samples Collection and Mononuclear Cells Isolation
			3.2 CD34+ Cell Isolation
			3.3 CD34+ Cells Cryopreservation
			3.4 CD34+ HSPCs Culture
			3.5 CD34+ HSPCs Transduction and Sorting
			3.6 Colony-Forming Unit Assay
			3.7 Cell Proliferation Assay
			3.8 Mice Transplantations and Follow-Up
			3.9 Analysis of Human Engraftment in  Mice
		4 Notes
		References
	Chapter 18: Lentiviral Transduction for Optimal LSC/HSC Manipulation
		1 Introduction
		2 Materials
			2.1 Lentiviral Preparation
			2.2 HSC Transduction
		3 Methods
			3.1 Transfection of 293T Cells for Viral Packaging/Concentration of Viral Supernatants
			3.2 Lentiviral Transduction of Purified Hematopoietic Stem Cells
		4 Notes
		References
	Chapter 19: Characterizing the In Vivo Role of Candidate Leukemia Stem Cell Genes
		1 Introduction
		2 Materials
			2.1 Virus Production
			2.2 Dissection and Isolation of Bone Marrow
			2.3 Viral Transduction
			2.4 Irradiation
			2.5 Intravenous Injections
			2.6 Peripheral Blood Analysis
			2.7 Postmortem Exam
		3 Methods
			3.1 Production of Retroviral Particles for AML Oncogenic Driver
			3.2 Production of Lentiviral Particles to Manipulate Expression of Candidate LSC Genes
			3.3 Preparation of Bone Marrow Samples for Transduction
			3.4 Viral Transduction of Lineage-Depleted Bone Marrow Cells
			3.5 Irradiation and Intravenous Injection into the Lateral Tail Vein of Recipient  Mice
			3.6 Collection of Peripheral Blood
			3.7 Analysis of Peripheral Blood by Hemocytometer and FACS
			3.8 Monitoring Disease Development
			3.9 Postmortem Examination of Spleen, Liver, and Bone Marrow of Diseased  Mice
		4 Notes
		References
	Chapter 20: Clonal Analysis of Patient-Derived Samples Using Cellular Barcodes
		1 Introduction
		2 Materials
			2.1 Transfection
			2.2 Transduction
			2.3 DNA Isolation
			2.4 PCR
			2.5 Agarose  Gel
			2.6 PCR Product Purification
			2.7 PCR Product Quality Control
		3 Methods
			3.1 Considerations for Barcode Library Production
			3.2 Transfection
				3.2.1 Day-7: Thaw HEK293FT Cells
				3.2.2 Day 0: Plate HEK293FT Cells
				3.2.3 Day 2: Transfection
				3.2.4 Day 3: Medium Change
				3.2.5 Day 4: Harvest Virus
			3.3 Transduction of a Cell Line: Quality Control of Produced Virus
				3.3.1 Day-7: Thaw SupB15 Cells
				3.3.2 Day-6: Refresh Culture Medium
				3.3.3 Day 0: Pre-coat Wells with RetroNectin
				3.3.4 Day 1: Transduction
				3.3.5 Day 2: Remove Virus
				3.3.6 Day 3: Determine Transduction Efficiency
			3.4 Transduction of Patient-Derived B-ALL Cells
				3.4.1 Day 0: Pre-coat Wells with RetroNectin
				3.4.2 Day 1: Thaw and Transduce Patient-Derived B-ALL Cells
				3.4.3 Day 2: Remove Virus
				3.4.4 Day 3: Determine Transduction Efficiency
			3.5 Barcode Retrieval by Next-Generation Sequencing
				3.5.1 Isolation of Genomic DNA
				3.5.2 Barcode Amplification by Standard PCR
				3.5.3 Barcode Amplification by Nested PCR
				3.5.4 PCR Product Cleanup of Pooled Samples
				3.5.5 Quality Control of Purified Barcode Sequences
			3.6 Data Processing
				3.6.1 Pre-filtering
				3.6.2 Check for Multiple Integrations per  Cell
				3.6.3 Removing Sequencing Noise
				3.6.4 Counting Clones
		4 Notes
		References
	Chapter 21: Arrayed Molecular Barcoding of Leukemic Stem Cells
		1 Introduction
		2 Materials
			2.1 Lentivirus Production
			2.2 Isolation of Leukemia Cells from Mice
				2.2.1 Isolation of c-Kit+ Cells from Bone Marrow
				2.2.2 Lentiviral Transduction and Titration
				2.2.3 Arrayed Screen
				2.2.4 In Vivo Readout of LSCs and Genomic DNA Isolation
				2.2.5 Preparation of Libraries and Sequencing
		3 Methods
			3.1 Production of Lentiviruses Containing Barcode Sequences
			3.2 Isolation of Leukemic Cells from Mice
			3.3 Test of Transduction Efficacy in Target Cells
			3.4 Performing Screens Using Cells Labeled with Arrayed Molecular Barcodes
				3.4.1 Barcoding of c-Kit+ Leukemia Cells
				3.4.2 Arrayed Ex Vivo Screening
				3.4.3 In Vivo Readout
			3.5 DNA Preparation and Sequencing
				3.5.1 Isolation of Genomic DNA
				3.5.2 PCR Amplification of Barcodes
				3.5.3 Indexing of the Samples
				3.5.4 Sequencing and Analysis
		4 Notes
		References
	Chapter 22: In Vivo Generation of Leukemic Stem Cells by HSC Targeting by Transgenesis
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Reagents
		3 Methods
			3.1 Preparation of the Transgene
				3.1.1 Expansion of the Plasmid Containing the Oncogene
				3.1.2 Restriction Enzyme Digestion
				3.1.3 Agarose Gel-Electrophoresis
				3.1.4 Gel Extraction
				3.1.5 Subcloning of the Oncogene cDNA into the Plasmid Containing the Promoter
				3.1.6 Expansion of the Transgene Plasmid Prior to Purification
				3.1.7 Transgene Purification
			3.2 Transgene Pronuclear Microinjection
			3.3 Genotyping and Analysis of Founders
		4 Notes
		References
	Chapter 23: In Situ Hematopoietic Stem Cell Imaging
		1 Introduction
		2 Materials
			2.1 Mouse Preparation
			2.2 Whole Mount Immunostaining
		3 Methods
			3.1 Preparation of Whole Mount Sternal Tissue
			3.2 Immunostaining of HSCs in Whole Mount Tissue
			3.3 Sample Orientation for Microscopy
			3.4 Microscopy
			3.5 Analysis
		4 Notes
		References
	Chapter 24: A Genome Editing System for Therapeutical Targeting of Stem Cells
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 RNP Preparation
			2.3 Electroporation
			2.4 Mice Transplantation and Sacrifice
			2.5 Antibodies
			2.6 PCR-Based Analysis of Editing Efficiency
		3 Methods
			3.1 Cell Thawing
			3.2 Cell Electroporation
			3.3 Xenotransplantation in Immunodeficient Mice
			3.4 Analysis of Engraftment and Multilineage Differentiation of Edited HSCs
			3.5 Measurement of Editing Frequency by Sanger Sequencing and TIDE Analysis
			3.6 Colony-Forming Cell Assay
		4 Notes
		References
	Chapter 25: Method for the Generation of Induced Hematopoietic Stem Cells
		1 Introduction
			1.1 Hematopoietic Stem Cells
			1.2 Strategies for Reprogramming into  HSCs
		2 Materials
			2.1 Mice
			2.2 Primary Hematopoietic Cell Sorting
			2.3 Cells
			2.4 Cloning
			2.5 Lentivirus Production
			2.6 Transplantation and Peripheral Blood Sampling upon Transplantation
		3 Methods
			3.1 Identification of Potential Candidate Genes for Reprogramming
			3.2 Cloning
			3.3 Production of Lentiviral Vectors
			3.4 Isolation of Primitive Hematopoietic Progenitors
			3.5 Transduction with Lentiviral Vectors
			3.6 Competitive Repopulation Assay
		References
	Chapter 26: Modeling Leukemia Stem Cells with Patient-Derived Induced Pluripotent Stem Cells
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Disposables
			2.3 Cells and Vectors
			2.4 Media Components and Reagents
			2.5 Reagent Setup
		3 Methods
			3.1 Reprogramming of BM/PB  MNCs
			3.2 Expansion of iPSCs and Establishment of Lines
			3.3 Cryopreservation of Cells
			3.4 Hematopoietic Differentiation
		4 Notes
		References
	Chapter 27: High-Content Imaging to Phenotype Human Primary and iPSC-Derived Cells
		1 Introduction
			1.1 Considerations, Technologies, Principles
			1.2 Benchmarking of Endothelial Cells and Extraction of Cells from Patients
		2 Materials
			2.1 hiPSC-EC Differentiation
			2.2 Isolation of Mononuclear Cells from Bone Marrow Samples
		3 Methods
			3.1 hiPSC-EC Differentiation
			3.2 Experiment Setup
			3.3 Cell Plating
			3.4 Cell Culture
			3.5 Cell Fixation
			3.6 Cell Immunostaining
			3.7 Image Segmentation
				3.7.1 Image Acquisition
				3.7.2 Find Nuclei
				3.7.3 Filter Image
				3.7.4 Find Cytoplasm
				3.7.5 Select Population
				3.7.6 Find Image
				3.7.7 Select Region
				3.7.8 Modify Population
				3.7.9 Find Spots
				3.7.10 Calculate Properties
				3.7.11 Select Population
				3.7.12 Define Results
			3.8 Multidimensional Reduction
			3.9 Isolation of Mononuclear Cells from Bone Marrow Samples
		4 Notes
			4.1 Notes for Cell Benchmarking Protocol
			4.2 Image Acquisition
			4.3 Image Segmentation
			4.4 Image Analysis
			4.5 Object Dimensions
			4.6 Texture Features
			4.7 Data Exploration and Visualization
			4.8 Data Transformation
			4.9 Data Modeling, Clustering, Classification, and Dynamics
		References
	Chapter 28: Bioinformatic Methods to Identify Mutational Signatures in Cancer
		1 Introduction
		2 Downloading the Input  Data
		3 Quick Start Guide for Mutational Signatures Analysis with SigProfilerExtractor
			3.1 Prerequisites
			3.2 Installing SigProfilerExtractor
			3.3 Installing a Reference Genome
			3.4 Performing Signatures Extraction
			3.5 Interpreting Signatures Extraction Results
		4 Detailed Protocol of Mutational Signature Extraction Using SigProfilerExtractor
			4.1 Software Implementation
			4.2 Methods and Materials to Extract Signatures Using Python Platform
				4.2.1 Required Operating System
				4.2.2 Required Tools
				4.2.3 Software Installation
				4.2.4 Data Preparation for Extracting Mutational Signatures
				Box 1 Description of SigProfilerExtractor´s Parameters in a Python Environment
				4.2.5 Extracting Mutational Signatures from a Matrix
				4.2.6 Extraction of Signatures from Mutational Catalogs
				4.2.7 Detailed Explanation of Results
		5 Methods and Materials to Extract Signatures Using R Platform
			5.1 Required Operating System
			5.2 Required Tools
			5.3 Software Installation
			5.4 Installation of Packages
			5.5 Data Preparation for Extracting Mutational Signatures
			Box 2 Description of SigProfilerExtractor´s Parameters in an R Environment
				5.5.1 Extraction of Signatures from Mutational Catalogs
		6 Notes
		References
Index