Name: identification of key factors regulating self-renewal and differentiation in eml hematopoietic precursor cells by rna-sequencing analysis
Uploaded: 2014-11-11T15:00:00
Description: Watch this Scientific Journal Video about Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis at JoVE.com

JQW, SZ, SD and KC are supported by grant from the National Institutes of Health and the Staman Ogilvie Fund&#8212;Memorial Hermann Foundation.

1. EML Cell Culture and Separation of Lin-CD34+ and Lin-CD34- Cells Using Magnetic Cell Sorting System and Fluorescence-activated Cell Sorting Method
<ol>
	<li>Preparation of baby hamster kidney (BHK) cell culture medium for stem cell factor collection:
	<ol>
		<li>Culture BHK cells in DMEM medium containing 10% FBS in 25 cm2 flask (Table 1) at 37 &#176;C, 5% CO2 in a cell culture incubator.</li>
		<li>When cells grow to 80 - 90% confluence, wash cells once with 10 ml of PBS. Add ...

<ol>
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Mammalian transcriptome is very complex34-38. RNA-Seq technology plays an increasingly important role in the studies of transcriptome analysis, novel transcripts detection and single nucleotide variation discovery etc. It has many advantages over other methods for gene expression analysis. As mentioned in the introduction, it overcomes the hybridization artifacts of microarray and can be used to identify novel transcripts de novo. One limitation of RNA-sequencing is relative short rea...

Hematopoietic stem cells are rare blood cells that reside mainly in the adult bone marrow niche. They are responsible for the production of cells required to replenish the blood and the immune systems1. As a kind of stem cells, HSCs are capable of both self-renewal and differentiation. Elucidating mechanisms that control the fate decision of HSCs, toward either self-renewal or differentiation, will offer valuable guidance on the manipulation of HSCs for blood disease researches and clinical usage2. One problem faced by the researchers is that HSCs can be maintained and expanded in vitro to a very limited extent; the vast majority of their progeny are partially differentiated in culture2.
In order to identify key regulators that control the processes of self-renewal and differentiation at a genome-wide scale, we used a mouse primitive hematopoietic progenitor cell line EML as a model system. This cell line was derived from murine bone marrow3,4. When fed with different growth factors, EML cells can differentiate into erythroid, myeloid, and lymphoid cells in vitro5. Importantly, this cell line can be propagated in large quantity in culture medium containing stem cell factor (SCF) and still retaining their multipotentiality. EML cells can be separated into subpopulations of self-renewing Lin-SCA+CD34+ and partially differentiated Lin-SCA-CD34- cells based on surface markers CD34 and SCA6. Similar to short-term HSCs, SCA+CD34+ cells are able of self-renewal. When treated with SCF, Lin-SCA+CD34+ cells can rapidly regenerate a mixed population of Lin-SCA+CD34+ and Lin-SCA-CD34- cells and continue to proliferate6. The two populations are similar in morphology and have similar levels of c-kit mRNA and protein6. Lin-SCA-CD34- cells are capable of propagating in media containing IL-3 instead of SCF3. Unveiling the key regulators in the EML cell fate decision will offer better understanding of cellular and molecular mechanisms in early developmental transition during hematopoiesis.
In order to investigate the underlying molecular differences between the self-renewing Lin-SCA+CD34+ and partially differentiated Lin-SCA-CD34- cells, we used RNA-Seq to identify differentially expressed genes. In particular, we focus on transcription factors, as transcription factors are crucial in determining cell fate. RNA-Seq is a recently developed approach that utilizes the capabilities of next-generation sequencing (NGS) technologies to profile and quantify RNAs transcribed from genome7,8. In brief, total RNA is poly-A selected and fragmented as the initial template.The RNA template is then converted into cDNA using reverse transcriptase. In order to map full-length RNA transcripts, using intact, non-degraded RNA for constructing cDNA library is important. For the purpose of sequencing, specific adapter sequences are added to both ends of cDNA. Then, in most cases, cDNA molecules are amplified by PCR and sequenced in a high-throughput manner.
After sequencing, the resulting reads can be aligned to a reference genome and a transcriptome database. The number of reads that map to the reference gene is counted and this information can be used to estimate the gene expression level. The reads can also be assembled de novo without a reference genome, enabling the study of transcriptomes in non-model organisms9. RNA-seq technology has also been used to detect splice isoforms10-12, novel transcripts13 and gene fusions14. In addition to the detection of protein-coding genes, RNA-Seq can also be used to detect novel and analyze transcription level of non-coding RNAs, such as long non-coding RNA15,16, microRNA17, siRNA etc.18. Because of the accuracy of this method, it has been utilized for detection of single nucleotide variations19,20.
Before the advent of RNA-Seq technology, microarray was the main method used for analyzing gene expression profile. Pre-designed probes are synthesized and subsequently attached to a solid surface to form a microarray slide21. mRNA is extracted and converted to cDNA. During the reverse transcription process, fluorescently labeled nucleotides are incorporated into the cDNA and the cDNA can be hybridized onto the microarray slides. The intensity of the signal collected from a specific spot depends on the amount of cDNA binding to the specific probe on that spot21. Compared with RNA-Seq technology, microarray has several limitations. First, microarray relies on the pre-existing knowledge of gene annotation, while RNA-Seq technology is able to detect novel transcripts at relative high background level, which limits its use when gene expression level is low. Besides, the RNA-Seq technology has much higher dynamic range of detection (8,000 fold)7, whereas, due to background and saturation of signals, the accuracy of microarray is limited for both highly and lowly expressed genes7,22. Finally, microarray probes differ in their hybridization efficiencies, which make the results less reliable when comparing relative expression levels of different transcripts within one sample23. Although RNA-Seq has many advantages over microarray, its data analysis is complex. This is one of the reasons that many researchers still use microarray instead of RNA-Seq. Various bioinformatics tools are required for RNA-Seq data processing and analysis24.
Among several next-generation sequencing (NGS) platforms, 454, Illumina, SOLID and Ion Torrent are the most widely used ones. 454 was the first commercial NGS platform. In contrast to the other sequencing platforms such as illumina and SOLID, the 454 platform generates longer read length (average 700 base reads)25. Longer reads are better for initial characterization of transcriptiome due to their higher assemble efficiency25. The main disadvantage of the 454 platform is its high cost per megabase of sequence. The Illumina and SOLID platforms generate reads with increased numbers and short lengths. The cost per megabase of sequence is much lower than the 454 platform. Due to the large numbers of short reads for the Illumina and SOLID platforms, data analysis is much more computationally intensive. The price of the instrument and reagents for sequencing for the Ion Torrent platform is cheaper and the sequencing time is shorter25. However, the error rate and the cost per megabase of sequence are higher compared to the Illumina and SOLID platforms. Different platforms have their own advantages and disadvantages and require different methods for data analysis. The platform should be chosen based on the sequencing purpose and the availability of funding.
In this paper, we take Illumina RNA-Seq platform as an example. We used EML cell as a model system to investigate the key regulators in EML cell self-renewal and differentiation, and provided a detailed methods of RNA-Seq library construction and data analysis for expression level calculation and novel transcript detection. We have shown in our previous publication that RNA-seq study in EML model system2, when coupled with functional test (e.g. shRNA knockdown) provide a powerful approach in understanding the molecular mechanism of the early stages of hematopoietic differentiation, and can serve as a model for the analysis of cell self-renewal and differentiation in general.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Invitrogen</td>
			<td>15240-062</td>
			<td>BHK cell culture</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD34 FITC</td>
			<td>eBioscience</td>
			<td>11-0341-81</td>
			<td>FACS sorting</td>
		</tr>
		<tr>
			<td>Anti-Mouse Ly-6A/E (Sca-1) PE</td>
			<td>eBioscience</td>
			<td>12-5981-81</td>
			<td>FACS sorting</td>
		</tr>
		<tr>
			<td>APC Mouse Lineage Antibody Cocktail</td>
			<td>BD Biosciences</td>
			<td>558074</td>
			<td>FACS sorting</td>
		</tr>
		<tr>
			<td>BD FACSAria Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>Special offer sysmtem</td>
			<td>FACS sorting</td>
		</tr>
		<tr>
			<td>Corning&#8482; Cell Culture Treated Flasks 75cm2</td>
			<td>Corning incorporated</td>
			<td>430641</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Corning&#8482; Cell Culture Treated Flasks 25cm2</td>
			<td>Corning incorporated</td>
			<td>430639</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I, Amplification Grade</td>
			<td>Invitrogen</td>
			<td>18068-015</td>
			<td>Library preparation</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>BHK cell culture</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>HI FBS&#160;</td>
			<td>Invitrogen</td>
			<td>16140071</td>
			<td>BHK cell culture</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Invitrogen</td>
			<td>16050-122</td>
			<td>EML cell culture</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>HyClone</td>
			<td>SH30228.02</td>
			<td>EML cell culture</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030-081</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Lineage Cell Depletion Kit, mouse&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-858</td>
			<td>Isolation of lineage negative cells</td>
		</tr>
		<tr>
			<td>NanoVue Plus spectrophotometer&#160;</td>
			<td>GE Healthcare</td>
			<td>28-9569-62</td>
			<td>Quality control</td>
		</tr>
		<tr>
			<td>Thermo Scientific&#8482; Napco&#8482; 8000 Water-Jacketed CO2 Incubators</td>
			<td>Thermo Scientific</td>
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			<td>Penicillin-Streptomycin</td>
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			<td>TruSeq&#8482; RNA Sample Prep Kit v2 -Set B (48rxn)</td>
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			<td>2100 Electrophoresis Bioanalyzer Instrument&#160;</td>
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identification of key factors regulating self-renewal and differentiation in eml hematopoietic precursor cells by rna-sequencing analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

In order to analyze differentially expressed genes in Lin-CD34+ and Lin-CD34- EML cells, we used RNA-Seq technology. Figure 1 shows the workflow of the procedures. After isolation of lineage negative cells by magnetic cell sorting, we separated Lin-SCA+CD34+ and Lin-SCA-CD34- cells using FACS Aria. Lin-enriched EML cells were stained with anti-CD34, anti-Sca1 and lineage cocktail antibodies. Only Lin- cells were gated for analysis of Sca1 and CD34 expression. Two populations (SCA+CD34+ and SCA-CD34- EML ...

Watch this Scientific Journal Video about Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis at JoVE.com

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis (Video) | JoVE

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

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