Name: genome-wide analysis of hdac inhibitor-mediated modulation of micrornas and mrnas in b cells induced to undergo class-switch dna recombination and plasma cell differentiation
Uploaded: 2017-09-20T12:30:00
Description: Watch this Scientific Journal Video about Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell

This work was supported by NIH grants AI 105813 and AI 079705 (to PC), the Alliance for Lupus Research Target Identification in Lupus Grant ALR 295955 (to PC), and the Arthritis National Research Foundation research grant (to HZ). TS was supported by the Pediatrics Medical Center, Second Xiangya Hospital, Central South University, Changsha, China, in the context of the Xiangya-UT School of Medicine San Antonio medical student visiting program.

The protocol follows the animal care guidelines of The Institutional Animal Care and Use Committees of the University of Texas Health Science Center at San Antonio.
1. Stimulation of Mouse B Cells for CSR, Plasma Cell Differentiation, and HDI Treatment
<ol>
	<li>Preparation of spleen cell suspensions 
	NOTE: Perform all steps in a laminar flow hood except for mouse euthanasia and dissection.
	<ol>
		<li>Euthanize the specific pathogen-free C57BL/6J mouse (8-12 weeks of ...

<ol>
	<li>Allis, C. D., Jenuwein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+hallmarks+of+epigenetic+control.">The molecular hallmarks of epigenetic control.</a> Nat Rev Genet. 17, 487-500 (2016).</li><li>Li, G., Zan, H., Xu, Z., Casali, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+of+the+antibody+response.">Epigenetics of the antibody response.</a> Trends Immunol. 34, 460-470 (2013).</li><li>Zan, H., Casali, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+of+Peripheral+B-Cell+Differentiation+and+the+Antibody+Response.">Epigenetics of Peripheral B-Cell Differentiation and the Antibody Response.</a> Front Immunol. 6, 631 (2015).</li><li>Xu, Z., Zan, H., Pone, E. J., Mai, T., Casali, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoglobulin+class-switch+DNA+recombination:+induction,+targeting+and+beyond.">Immunoglobulin class-switch DNA recombination: induction, targeting and beyond.</a> Nat. Rev. Immunol. 12, 517-531 (2012).</li><li>Nutt, S. L., Hodgkin, P. D., Tarlinton, D. M., Corcoran, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+generation+of+antibody-secreting+plasma+cells.">The generation of antibody-secreting plasma cells.</a> Nat. Rev. Immunol. 15, 160-171 (2015).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+target+recognition+and+regulatory+functions.">MicroRNAs: target recognition and regulatory functions.</a> Cell. 136, 215-233 (2009).</li><li>Fabian, M. R., Sonenberg, N., Filipowicz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mRNA+translation+and+stability+by+microRNAs.">Regulation of mRNA translation and stability by microRNAs.</a> Annu. Rev. Biochem. 79, 351-379 (2010).</li><li>White, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+inhibitors+upregulate+B+cell+microRNAs+that+silence+AID+and+Blimp-1+expression+for+epigenetic+modulation+of+antibody+and+autoantibody+responses.">Histone deacetylase inhibitors upregulate B cell microRNAs that silence AID and Blimp-1 expression for epigenetic modulation of antibody and autoantibody responses.</a> J Immunol. 193, 5933-5950 (2014).</li><li>Farh, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+epigenetic+fine+mapping+of+causal+autoimmune+disease+variants.">Genetic and epigenetic fine mapping of causal autoimmune disease variants.</a> Nature. 518, 337-343 (2015).</li><li>Nagalakshmi, U., Waern, K., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+method+for+comprehensive+transcriptome+analysis.">RNA-Seq: a method for comprehensive transcriptome analysis.</a> Curr Protoc Mol Biol. , 11-13 (2010).</li><li>Martin, J. A., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+transcriptome+assembly.">Next-generation transcriptome assembly.</a> Nat Rev Genet. 12, 671-682 (2011).</li><li>Luo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+expression+analysis+using+the+Illumina+microRNA-Seq+Platform.">MicroRNA expression analysis using the Illumina microRNA-Seq Platform.</a> Methods Mol. Biol. 822, 183-188 (2012).</li><li>Shen, T., Sanchez, H. N., Zan, H., Casali, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+reveals+selective+modulation+of+microRNAs+and+mRNAs+by+histone+deacetylase+inhibitor+in+B+cells+induced+to+uUndergo+class-switch+DNA+recombination+and+plasma+cell+differentiation.">Genome-wide analysis reveals selective modulation of microRNAs and mRNAs by histone deacetylase inhibitor in B cells induced to uUndergo class-switch DNA recombination and plasma cell differentiation.</a> Front. Immunol. 6, 627 (2015).</li><li>Trapnell, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+gene+and+transcript+expression+analysis+of+RNA-seq+experiments+with+TopHat+and+Cufflinks.">Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.</a> Nat Protoc. 7, 562-578 (2012).</li><li>Kin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fRNAdb:+a+platform+for+mining/annotating+functional+RNA+candidates+from+non-coding+RNA+sequences.">fRNAdb: a platform for mining/annotating functional RNA candidates from non-coding RNA sequences.</a> Nucleic Acids Res. 35, D145-D148 (2007).</li><li>Agarwal, V., Bell, G. W., Nam, J. W., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+effective+microRNA+target+sites+in+mammalian+mRNAs.">Predicting effective microRNA target sites in mammalian mRNAs.</a> Elife. 4, (2015).</li><li>Griffiths-Jones, S., Saini, H. K., van Dongen, S., Enright, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRBase:+tools+for+microRNA+genomics.">miRBase: tools for microRNA genomics.</a> Nucleic Acids Res. 36, D154-D158 (2008).</li><li>Anders, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=doRiNA:+a+database+of+RNA+interactions+in+post-transcriptional+regulation.">doRiNA: a database of RNA interactions in post-transcriptional regulation.</a> Nucleic Acids Res. 40, D180-D186 (2012).</li></ol>

This protocol provides comprehensive approaches to induce B cell class switching and plasma cell differentiation; to analyze their impact by epigenetic modulators, namely HDI; and to detect the effect of HDI on mRNA and miRNA expression in these cells. Most of these approaches can also be used to analyze the impact of epigenetic factor on human B-cell function and mRNA/miRNA expression. The qRT-PCR and mRNA-seq/miRNA-seq approaches can also be used to analyze B cells isolated from mice treated with epigenetic modulators,...

Epigenetic marks or factors, such as DNA methylation, histone posttranslational modifications, and non-coding RNAs (including microRNAs), modulate cell function by altering gene expression1. Epigenetic modifications regulate B lymphocyte function, such as immunoglobulin class-switch DNA recombination (CSR), somatic hypermutation (SHM), and differentiation to memory B cells or plasma cells, thereby modulating the antibody and autoantibody responses2,3. CSR and SHM critically require activation-induced cytidine deaminase (AID, encoded as Aicda), which is highly induced in B cells in response to T-dependent and T-independent antigens4. Class-switched/hypermutated B cells further differentiate into plasma cells, which secrete large volumes of antibodies in a fashion critically dependent upon B lymphocyte-induced maturation protein 1 (Blimp1, encoded as Prdm1)5. Abnormal epigenetic changes in B cells may result in aberrant antibody/autoantibody responses, which can lead to allergic response or autoimmunity1,4. Understanding how epigenetic factors, such as miRNAs, modulate B cell-intrinsic gene expression is not only important for vaccine development, but is also essential to reveal the mechanisms of potential abnormal antibody/autoantibody responses.
Histone acetylation and deacetylation are modifications of the lysine residues on histone proteins typically catalyzed by histone acetyltransferase (HAT) and histone deacetylase (HDAC). These modifications lead to the increasing or decreasing accessibility of chromatin and further allow or prevent the binding of transcription factors or proteins to DNA and the alteration of gene expression5,6,7,8. HDAC inhibitors (HDI) are a class of compounds that interfere with the function of HDACs. Here, we used HDI (VPA) to address the regulation of HDAC on the intrinsic gene expression profile of B cells and on its mechanism.
miRNAs are small, non-coding RNAs approximately 18 to 22 nucleotides in length that are generated through several stages. miRNA host genes are transcribed and form hairpin primary microRNAs (pri-miRNAs). They are exported to the cytoplasm, where pri-miRNAs are further processed into precursor miRNAs (pre-miRNAs). Finally, mature miRNAs are formed through the cleavage of the pre-miRNAs. miRNAs recognize the complementary sequences within the 3&#39; untranslated region of their target mRNAs6,7. Through post-transcriptional silencing, miRNAs regulate cellular activity, such as proliferation, differentiation, and apoptosis10,11. Since multiple miRNAs can target the same mRNA, and one single miRNA can potentially target multiple mRNAs, it is important to have an in-context view of the miRNA expression profile to understand the value of the individual and the collective effect of miRNAs. miRNAs have been shown to be involved in B-cell development and peripheral differentiation, as well as B-cell stage-specific differentiation, antibody response, and autoimmunity1,4,9. In the 3&#39; UTR of Aicda and Prdm1, there are several validated or predicted evolutionarily conserved sites that can be targeted by miRNAs8.
Epigenetic modulation, including histone post-transcription modification and miRNAs, display a cell-type and cell stage-specific regulation pattern of gene expression9. Here, we describe methods to define the HDI-mediated modulation of miRNA and mRNA expression, CSR, and plasma cell differentiation. These include protocols for inducing B cells to undergo CSR and plasma cell differentiation; for treating the B cells with HDI; and for analyzing miRNA and mRNA expression by qRT-PCR, miRNA-seq, and mRNA-seq10,11,12,8,13.

<table border="0" cellpadding="0" cellspacing="0" width="854">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:528px;">C57BL/6 mice</td>
			<td style="width:152px;">Jackson Labs</td>
			<td align="right" style="width:111px;">664</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning cellgro RPMI 1640 Medium (Mod.) 1X with L-Glutamine (Size: 6 x 500mL; With L-Glutamine)</td>
			<td>Fisher Scientific</td>
			<td>MT 10-040-CV&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Hyclone</td>
			<td>SH300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HyClone Antibiotic Antimycotic Solution 100 mL</td>
			<td>Fisher Scientific - Hyclone</td>
			<td>SV3007901&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-Mercaptoethanol</td>
			<td>Fisher Scientific</td>
			<td>44-420-3250ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Cell Strainers</td>
			<td>Fisher Scientific</td>
			<td>21008-952&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Stain 0.04%</td>
			<td>GIBCO/Life Technologies/Inv</td>
			<td align="right">15250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACK Lysis Buffer&#160;</td>
			<td>Fisher Scientific</td>
			<td>BW10-548E&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hausser Scientific Bright-Line Counting Chamber</td>
			<td>Fisher Scientific</td>
			<td>02-671-51B</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:528px;">EasySep Magnet</td>
			<td>Stem Cell Technologies</td>
			<td align="right">18000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Round-Bottom Polystyrene Tubes with Cap</td>
			<td>Fisher Scientific</td>
			<td>14-959-1A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EasySep Mouse B cell Isolation Kit</td>
			<td>Stem Cell Tech</td>
			<td align="right">19854</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Needle Only 18 Gauge 1.5 inch SHORT BEVEL 100/box&#160;</td>
			<td>BD Biosciences</td>
			<td>&#160;305199</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE/Cy7 anti-mouse CD138 (Syndecan-1) Antibody</td>
			<td>BioLegend&#160;</td>
			<td>142513 (25 ug)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE-Cy7 B220 antibody</td>
			<td>BioLegend</td>
			<td align="right">103222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7-AAD (1 mg)</td>
			<td>Sigma Aldrich</td>
			<td>A9400-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APC anti-mouse/human CD45R/B220 antibody</td>
			<td>Biolegend</td>
			<td align="right">103212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse APC-IgG1 200 &#181;g</td>
			<td>Biolegend</td>
			<td align="right">406610</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC anti-mouse IgM Antibody</td>
			<td>Biolegend</td>
			<td align="right">406506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC anti-mouse/human CD45R/B220 Antibody</td>
			<td>Biolegend</td>
			<td align="right">103206</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE Anti-Human/Mouse CD45R (B220) (RA3-6B2)</td>
			<td>Biolegend</td>
			<td align="right">103208</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS 1X</td>
			<td>Fisher Scientific</td>
			<td>MT-21-022-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin, Fraction V, Heat Shock Treated&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LPS 25mg (Lipopolysaccharides from Escherichia coli 055:B5)</td>
			<td>Sigma Aldrich</td>
			<td>L2880-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant mouse IL-4 (carrier-free)</td>
			<td>BioLegend</td>
			<td>574302 (size: 10 ug)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Valproic acid sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>P4543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SterilGARD e3 Class II Type A2 Biosafety Cabinet</td>
			<td>The Baker Company</td>
			<td>SG404</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Large-Capacity Reach-In CO2 Incubator&#160;</td>
			<td>Thermo Scientific</td>
			<td align="right">3950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isotemp Digital-Control Water Baths: Model 205</td>
			<td>Fisher Scientific</td>
			<td>15-462-5Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5mL Round Bottom Polystyrene Test Tube</td>
			<td>Fisher Scientific</td>
			<td>&#160;14-959-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning CentriStar 15ml Centrifuge Tubes&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>05-538-59A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.7 mL Microtube, clear</td>
			<td>Genesee</td>
			<td>22-282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Higher-Speed Easy Reader Plastic Centrifuge Tubes 50ml</td>
			<td>Fisher Scientific</td>
			<td>06-443-18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ELMI SkySpin CM-6MT</td>
			<td>ELMI</td>
			<td>CM-6MT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotor 6M</td>
			<td>ELMI</td>
			<td>6M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotor 6M.06</td>
			<td>ELMI</td>
			<td>6M.06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drummond Portable Pipet-Aid XP Pipet Controller</td>
			<td>Drummond Scientific</td>
			<td>4-000-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 mL serological pipette tips</td>
			<td>Fisher Scientific</td>
			<td>89130-900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL serological pipette tips</td>
			<td>Fisher Scientific</td>
			<td>89130-898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 mL serological pipette tips</td>
			<td>Fisher Scientific</td>
			<td>898130-896</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">48-well plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-86</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Allegra 6 Benchtop Centrifuge, Non-Refrigerated</td>
			<td>Beckman Coulter</td>
			<td align="right">366802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GH-3.8A Rotor, Horizontal, ARIES Smart Balance</td>
			<td>Beckman Coulter</td>
			<td align="right">366650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Allegra 25R Benchtop Centrifuge, Refrigerated</td>
			<td>Beckman Coulter</td>
			<td align="right">369434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TA-15-1.5 Rotor, Fixed Angle</td>
			<td>Beckman Coulter</td>
			<td align="right">368298</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisher Scientific AccuSpin Micro 17</td>
			<td>Fisher Scientific</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisher Scientific Analog Vortex Mixer&#160;</td>
			<td>Fisher Scientific</td>
			<td>02-215-365</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">miRNeasy Mini Kit (50)</td>
			<td>Qiagen</td>
			<td align="right">217004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Direct-zol RNA MiniPrep kit</td>
			<td>Zymo Research</td>
			<td>R2050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform (Approx. 0.75% Ethanol as Preservative/Molecular Biology)</td>
			<td>Fisher Scientific</td>
			<td>BP1145-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rnase-Free Dnase set (50)</td>
			<td>QIAGEN</td>
			<td align="right">79254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop 2000 Spectrophotometers</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superscript III First-strand Synthesis System RT-PCR</td>
			<td>Invitrogen</td>
			<td align="right">175013897</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iTaq Universal SYBR Green Supermix</td>
			<td>Bio-rad&#160;</td>
			<td>172-5121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand 96-Well Semi-Skirted PCR Plates, case of 25</td>
			<td>Fisher</td>
			<td style="width:111px;">14-230-244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microseal &#39;B&#39; Adhesive Seals</td>
			<td>Bio-Rad</td>
			<td>MSB-1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MyiQ Optics Module</td>
			<td>Bio-Rad</td>
			<td>170-9744</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iCycler Chassis</td>
			<td>Bio-Rad</td>
			<td>170-8701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optical Kit</td>
			<td>Bio-Rad</td>
			<td>170-9752</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD LSR II Flow Cytometry Analyzer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSDiva software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo 10</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2100 Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2943CA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S200 Focused-ultrasonicator</td>
			<td>Covaris</td>
			<td>S200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPRIworks Fragment Library System I for Illumina</td>
			<td>Beckman Coulter</td>
			<td>A288267</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cBot Cluster Generation Station</td>
			<td>illumina</td>
			<td>SY-312-2001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HiSeq 2000 Genome Sequencer</td>
			<td>Illumina</td>
			<td>SY-401-1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq RNA Library Prep Kit v2</td>
			<td>Illumina</td>
			<td>RS-122-2001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruSeq Small RNA Library Prep Kit</td>
			<td>Illumina</td>
			<td>RS-200-0012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NEXTflex Illumina Small RNA Sequencing Kit v3</td>
			<td>Bioo Scientific</td>
			<td>5132-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2200 TapeStation</td>
			<td>Agilent</td>
			<td>G2964AA</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome-wide analysis of hdac inhibitor-mediated modulation of micrornas and mrnas in b cells induced to undergo class-switch dna recombination and plasma cell differentiation

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Using our protocol, purified B cells placed with LPS (3 &#181;g/mL) and IL-4 (5 ng/mL) for 96 h can induce 30-40% of CSR to IgG1 and ~10% of plasma cell differentiation. After treatment with HDI (500&#181;M VPA), the CSR to IgG1 decreased to 10-20%, while plasma cell differentiation decreased to ~2% (Figure 1). HDI-mediated inhibition of CSR was further confirmed by decreased numbers of post-recombination I&#956;-C&#947;1 and mature VHDJH-C&#947;1 tr...

Watch this Scientific Journal Video about Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

Research

JoVE Journal

Immunology and Infection

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

An In Vivo Mouse Model to Measure NaÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¯ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...