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

The overall goal of this procedure is to identify potential key regulators of mouse EML cell self-renewal and differentiation by using RNA sequencing technology. This is accomplished by first separating lineage negative CD 34 positive and lineage negative CD 34 negative EML cells based on surface markers. The second step is to isolate mRNA, convert the mRNA into cDNA and prepare the library for sequencing.

Next, the DNA library is subjected to high throughput sequencing. The final step is to analyze the sequencing results and look for differentially expressed transcription factors. Ultimately, RNA sequencing technology is used to show differentially expressed transcription factors in lineage negative CD 34 positive and lineage negative CD 34 negative EML cells.

The main advantage of RNA sequencing over other G expression prolene methods such as microray, is that it has higher sensitivity, accuracy and wider dynamic range, and it does not rely on preexisting gene annotation information. This method can help to answer the key questions in the stem cell field, such as what are the key transcript factors regulating cell self renewal and differentiation. The implication of this technique extend to where the therapy of blood disease because better understanding of key regulator controlling the hematopoietic precursor cells, self renewal and differentiation can lead to the developments of effective treatments.

Generally, individual new to this method will struggle because various bioformatics tools are required for the sequencing analysis. The baby hamster kidney or BHK cells for stem cell factor collection are cultured in dmem medium containing 10%FBS in a 25 square centimeter, flask at 37 degrees Celsius and 5%CO2. When the cells have reached 80 to 90%confluence trypsin eyes and collect them by centrifugation at 200 Gs for five minutes at room temperature, discard the snat and resuspend the cell palette in 10 milliliters of fresh BHK cell culture medium.

Transfer two milliliters of the cell suspension to a new 75 square centimeter flask an app and add 48 milliliters of fresh BHK cell culture. Medium to the flask culture. The BHK cells for two days.

After two days, collect the culture medium and passage it through a point 45 micrometer filter. Store the medium at negative 20 degrees Celsius. The mouse erythroid, myeloid, and lymphocytic or EML cells used in this protocol are cultured in suspension in EML basic medium containing BHK cell culture medium at 37 degrees Celsius and 5%CO2.

Maintain the EML cells at a low cell density with a peak density of fewer than 600, 000 cells per milliliter. Split the cells every two to three days at a ratio of one to five passage to cells gently, but for no more than 10 generations to deplete lineage positive cells. Harvest the EML cells by centrifugation at 200 Gs for five minutes.

Wash the cells once with PBS and centrifuge again after discarding the supernatant. Re suspend the cells in PBS and count the cells with a hemo cytometer. The number of cells is needed for determining the antibody concentration in the subsequent step.

Isolate the lineage negative or line negative cells using a lineage antibody cocktail and a magnetic activated cell sorting system according to the manufacturer's instructions. To begin this procedure, spin down the lin negative cells at 200 Gs for five minutes. Resuspend the cell pellet with PBS and count the cells with a hemo cytometer.

Wash the cells twice with fax buffer. After the second wash, pellet the cells at 200 Gs for five minutes. Label the five 1.5 milliliter micro centrifuge tubes with the numbers 1, 2, 3, 4, and five respectively.

Resuspend the cells with an appropriate volume of facts buffer to a concentration of 1 million cells per 100 microliters. Then aliquot 100 microliters of cell suspension to each of the five micro centrifuge tubes. Add one microgram of anti mouse CD 34 FE antibody to tube one and tube two.

Mix the tubes gently incubate all tubes at four degrees Celsius for one hour in the dark. Next, add point 25 micrograms of PE conjugated antica, one antibody and 20 microliters of a PC conjugated lineage. Cocktail antibodies to two one, add point 25 micrograms of PE conjugated antica one antibody to tube three and 20 microliters of a PC conjugated lineage.

Cocktail antibodies to tube four. Mix the tubes gently and incubate the cells at four degrees Celsius for an additional 30 minutes in the dark. After 30 minutes, add 300 microliters of fax buffer to all tubes and spin down the cells at 200 Gs for five minutes.

Wash the cells three times with 500 microliters of fax buffer after the final wash. Resus suspend each cell pellet in 500 microliters of fax buffer isolate lin negative SCA positive CD 34 positive and lin negative SCA negative CD 34 negative cells in tube one by fluorescence activated cell sorting. Total RNA is extracted from the lin negative CD 34 positive and lin negative CD 34 negative cells using triol and contaminated DNA is removed using deoxyribo nuclease one.

Following the manufacturer's protocol, assess the quality of the total RNA using a bioanalyzer following the manufacturer's instructions only RNA samples with an RNA integrity number or RIN of greater than nine are used for mRNA extraction and subsequent library construction procedures. This figure shows the result of a high quality RNA sample with an RIN equal to 9.4. The library construction protocol used here is specific to the Illumina RNA sequencing platform and utilizes an RNA sample preparation kit.

Follow the instructions provided by the manufacturer because only selected steps are shown in this video. Begin by positively selecting poly MNA using oligo DT magnetic beads. Next, fragment the poly mRNA perform reverse transcription using random primers to convert the mRNA to CD NA.Subsequently, the synthesized second strand CD NA is used to generate double stranded DNA.

Next, the three prime overhangs are removed and the five prime overhangs are filled by DNA polymerase denate. The three prime ends to CD NA fragments from ligating to one another. Add multiplex indexing adapters to both ends of the double stranded CD NA for sequencing.

Finally, perform PCR for the enrichment of the CD NA for fragments using a spectrophotometer measure the absorbance two 60 over absorbance two 80 of the amplified DNA. To obtain information about the concentration of the library, assess the library quality and measure the size range of the DNA fragments using a bioanalyzer. The methods for RNA sequencing data analysis are found in the accompanying protocol Text and include data file processing for downstream analysis, detecting novel transcripts and evaluating the expression level detecting differentially expressed genes and identifying differentially expressed transcription factors.

Lineage negative EML cells were stained with anti CD 34 antica one and lineage cocktail antibodies and lin negative skull positive CD 34 positive and lin negative sca. Negative CD 34 negative cells were separated using fluorescent activated cell sorting only. Lin negative cells were gated for analysis of SCA one and CD 34 expression.

Two populations of EML cells were observed when library construction was finished. The size of the DNA fragments in the library was checked using a bioanalyzer. This representative data shows a good quality library, but the fragment size peaking at about 300 base pairs.

The D-E-S-E-Q algorithm was used for differential expression analysis with a focus on differentially expressed transcription factors because transcription factors are crucial for sulfate determination. Transcription factors that change more than 1.5 fold between lin negative CD 34 positive and lin negative CD 34 negative were found and are shown in this heat map. Notably, the relative expression level of TCF seven in lin negative CD 34 positive cells is more than 100 fold higher than that in lin negative CD 34 negative cells.

Thus TCF seven was chosen for further chromatin immunoprecipitation and sequencing analysis and functional testing to confirm its function in the regulation of self-renew and differentiation in EML cells. Once master the RNA library can be finished in a few days and the entire procedure can be complete in about a month if it is performed properly After its development. This technology paved the way for researchers who are willing to explore differentially gene expression among diverse cell types and tissues such as B cells and malignant B cells.

While attempting this procedure, it is important to remember to use high CAR TRNA because it's the basis of the whole RNA sick procedure. After watching this video, you should have a better understanding of how to identify key regulators controlling EML cell cell renewal and differentiation.