这项工作得到了台湾国家科学技术委员会的支持。[NSTC 111-2314-B-006-003]

1. Poly（A） RNA纯化
注：在进行poly（A） RNA纯化之前，使用用DNase I处理的总RNA，并通过毛细管或常规凝胶电泳评估检查总RNA的质量和完整性。研究人员应该能够识别高分子量场中的 28S 和 18S rRNA 核糖体条带以及低分子量场中的 5.8S rRNA 带，而电泳图中没有任何明显的拖尾带。纯化步骤基本上遵循制造商的说明，并在特定步骤中进行了细微的修改。有关本协议中使用的所有材料和仪器的详细信息，请参阅 材料表 。
<ol><li>制备<ol><li>在进行以下程序之前，在室温下预热试剂、洗涤缓冲液、oligo（dT）珠子和裂解缓冲液30分钟。</li>
		<li>为了分离富集poly（A）的RNA，通过将足量的RNA转移到无核酸酶的1.5mL微量离心管中，制备10-20μg高质量总RNA的等分试样作为起始原料。在 70°C 的加热块中孵育5分钟，然后在冰上保持2 分钟。 注：作为起始材料的总RNA的量应根据所需的mRNA量和所用细胞的性质进行适当调整。根据经验，富集poly（A）的RNA可能占总RNA丰度的1-5%。10 μg 高质量总 RNA 的等分试样应提供 100-500 ng 范围内的高质量富集 poly（A） RNA，用于下游亚硫酸氢盐转化。</li>
		<li>同时，完全重悬oligo（dT）磁珠。将所需量的珠子悬浮液转移到新的 1.5 mL 微量离心管中，然后放在磁铁上 3 分钟，直到磁珠形成沉淀。 注：使用 50 μL oligo（dT） 磁珠处理 10 μg 总 RNA 起始量。根据所使用的细胞，可以进一步优化此步骤中的磁珠体积。</li>
		<li>除去上清液，用等量（步骤1.1.3中使用的珠子悬浮液的体积）重悬寡核苷酸（dT）珠子，并在除去上清液之前置于磁铁上3分钟。 注意： 不要过度干燥珠子;用户应快速继续执行步骤 1.2.1。</li>
	</ol></li>
	<li>第一轮净化<ol><li>将裂解缓冲液加入 RNA 样品管（体积比 4：1）并充分混合，然后转移到 oligo（dT） 磁珠中。 注：如果 RNA 样品体积为 20 μL，则加入 80 μL 裂解缓冲液。</li>
		<li>通过移液至少 10 次，将裂解缓冲液混合物和 oligo（dT） 微球完全重悬。</li>
		<li>让样品在室温下连续旋转孵育15分钟。</li>
		<li>将样品放在磁铁上5分钟或直到上清液澄清;弃去上清液。</li>
		<li>向珠子中加入 600 μL 洗涤缓冲液 1，小心混合以重悬珠子。</li>
		<li>将试管放在磁铁上5分钟或直到上清液澄清，弃去上清液，重复洗涤步骤1.2.5一次。</li>
		<li>加入 300 μL 洗涤缓冲液 2 洗涤珠子，并轻轻重悬珠子。</li>
		<li>将试管放在磁铁上5分钟或直到上清液澄清，弃去上清液，重复洗涤步骤1.2.7一次。</li>
		<li>向样品管中加入30μL冷的10mM Tris-HCl（洗脱缓冲液），并在 70°C 下孵育 5分钟。</li>
		<li>快速将试管转移到磁铁上，当悬浮液在20秒或更长时间后澄清时，将含有洗脱的poly（A）富集RNA的上清液转移到新的1.5mL微量离心管中。</li>
	</ol></li>
	<li>第二轮净化<ol><li>向洗脱的RNA样品中加入120μL（洗脱样品体积的4倍）裂解缓冲液并充分混合。</li>
		<li>用等量的裂解缓冲液洗涤第一轮纯化中使用的珠子，如步骤1.1.4，移液10次以重悬珠子，然后将它们放在磁铁上5分钟，然后弃去上清液。</li>
		<li>将RNA：裂解缓冲液混合物转移到洗涤的珠子中，并通过移液至少10x彻底混合。</li>
		<li>将样品在室温下连续旋转孵育15分钟。</li>
		<li>重复步骤1.2.4至1.2.8以除去盐和其他RNA亚型。 注：洗涤缓冲液的体积可减少至 300 μL 洗涤缓冲液 1 和 150 μL 洗涤缓冲液 2。</li>
		<li>向样品管中加入25μL（所需体积）冷的10mM Tris-HCl（洗脱缓冲液），并在 70°C 下孵育 5分钟。</li>
		<li>快速将试管转移到磁铁上，当悬浮液在20秒或更长时间后澄清时，将含有洗脱的poly（A）富集RNA的上清液转移到新的1.5mL微量离心管中。</li>
		<li>将 2.2 μL 转移到 8 联管中，使用 RNA 高灵敏度测定法对 RNA 数量和质量进行质量评估。</li>
		<li>将样品在-20°C下短期储存，在-70°C下储存较长时间。 注意：这可以是协议中的暂停点。</li>
	</ol></li>
</ol>2.亚硫酸氢盐转化
注：离心步骤均在室温下进行。这些程序基本上是按照制造商的说明执行的，但有一个额外的步骤 2.2，用于在亚硫酸氢盐反应步骤 2.3 之前添加加标对照 mRNA 序列。
<ol><li>将 19 μL 纯化的富集 poly（A） RNA 样品转移到 0.2 mL 8 联管中。</li>
	<li>加入1.0μL加标对照，其以预定的1：10,000数量比（即，0.1ng荧光素酶mRNA：1μg纯化的poly（A）富集RNA）无任何修饰）到步骤2.1的RNA样品中。 注：加标对照序列可以是萤火虫荧光素酶RNA、肾荧光素酶或不含甲基化胞嘧啶的 体外 转录RNA序列。</li>
	<li>向试管中加入 130 μL 转化摄政剂，轻轻倒置数次以充分混合。</li>
	<li>将试管放入PCR机中，在70°C下加热10分钟，然后在64°C下孵育45分钟，然后最后一步冷却至4°C。</li>
	<li>将色谱柱放在收集管上，将 250 μL RNA 结合缓冲液转移到色谱柱上。</li>
	<li>将步骤2.4中的样品转移到步骤2.5中的色谱柱中，并彻底移液。</li>
	<li>向色谱柱中加入 400 μL 95-100% 乙醇，迅速盖上盖子，倒置数次。</li>
	<li>在25°C下以10,000× g 离心30秒，弃去流出物。</li>
	<li>向色谱柱中加入 200 μL RNA 洗涤缓冲液。</li>
	<li>在25°C下以10,000× g 离心30秒，弃去流出物。</li>
	<li>加入 200 μL 脱磺缓冲液，在室温下孵育 30 分钟。</li>
	<li>在25°C下以10,000× g 离心30秒，弃去流出物。</li>
	<li>加入400μLRNA洗涤缓冲液，在25°C下以10,000× g 离心30秒，弃去流出物。重复此步骤一次。</li>
	<li>在25°C下再次以10,000× g 离心2分钟，以完全除去残留的洗涤缓冲液。 注：洗脱前的另一个离心步骤2.14 2分钟是完全除去洗涤缓冲液。洗涤步骤执行两次，以洗掉色谱柱上亚硫酸氢盐试剂和脱磺缓冲液中的高盐组分。</li>
	<li>将色谱柱转移到新的 1.5 mL 微量离心管中。</li>
	<li>向色谱柱中加入 20-30 μL 无核酸酶的 H2O，并在室温下静置 1 分钟。</li>
	<li>在25°C下以10,000× g 离心30秒。</li>
	<li>将 2.2 μL 转移到 8 条管中，以评估 RNA 的数量和质量。</li>
	<li>将亚硫酸氢盐处理的RNA样品在-20°C下短期储存，在-70°C下储存较长时间。 注意：这可以是协议中的暂停点。</li>
</ol>3. 亚硫酸氢盐处理的mRNA文库制备
注：按照文库制备说明方案第 4 部分用于纯化的 mRNA 或 rRNA 耗尽的 RNA。第一个启动步骤应遵循 FFPE RNA 方案，因为亚硫酸氢盐处理会使 RNA 片段化。在层流罩中执行每一步，并将反应混合物添加到冰冷冷却架上。
<ol><li>引发起始RNA<ol><li>转移所需量的亚硫酸氢盐处理的 mRNA 样品，总量最大为 5 μL，或加入无核酸酶的 H2O 使总量为 5 μL。 注：建议将至少 10 ng 亚硫酸氢盐处理的 mRNA 作为输入，用于此类 RNA 文库制备并生成 4nM 文库产物的最终摩尔浓度。</li>
		<li>向样品中加入 1 μL 随机引物（丁香），并通过移液混合。</li>
		<li>将样品管置于预设在65°C的PCR机中，并在105°C下盖上盖子5分钟，并在4°C下保持。</li>
	</ol></li>
	<li>第一链cDNA合成<ol><li>向样品中加入 4 μL 合成反应缓冲液（淡紫色）、8 μL 链特异性试剂（棕色）和 2 μL 合成酶混合物（棕色），总体积为 20 μL。 通过移液至少 10 倍并快速旋转彻底混合。</li>
		<li>将样品管置于预设的PCR机中，在25°C下10分钟，42°C下50分钟，70°C下15分钟，并在4°C下保持。 注：对于超过200个碱基的插入物，建议42°C孵育期为50分钟;对于低于200个碱基的插入片段，建议在42°C孵育15分钟。</li>
	</ol></li>
	<li>第二链cDNA合成<ol><li>加入 8 μL 含有 dUTP 混合物（橙色）、4 μL 合成酶混合物（橙色）和 48 μL 无核酸酶 H2O 的合成反应缓冲液，通过移液至少 10 次彻底混合并快速旋转。</li>
		<li>将样品管置于预设在16°C的PCR机中1小时，盖子关闭或≤40°C。</li>
	</ol></li>
	<li>用DNA纯化微球纯化双链cDNA<ol><li>在进行纯化前预热DNA纯化珠30分钟，并涡旋以重悬珠子。</li>
		<li>从步骤3.3.2中向cDNA样品中加入144μL重悬的DNA纯化珠，并通过移液至少10x充分混合。</li>
		<li>在室温下孵育5分钟。</li>
		<li>将样品管放在磁性架上5分钟，直到溶液澄清，然后除去上清液并保留含有DNA的珠子。</li>
		<li>将 200 μL 新鲜制备的 80% 乙醇加入带有珠子的试管中，同时将样品保持在磁架上并在室温下孵育 30 秒。</li>
		<li>除去上清液并重复步骤3.4.5一次，但这次小心地除去所有残留的上清液。</li>
		<li>将珠子风干 1-5 分钟，同时将样品保持在磁铁上。 注意： 不要过度干燥珠子;颜色应为深棕色。</li>
		<li>从磁架中取出样品，并通过向磁珠中加入 53 μL 0.1x TE（10 mM Tris-HCl 和 1 mM EDTA，pH 8.0）缓冲液来洗脱 DNA。通过移液至少10次彻底混合，并在室温下孵育2分钟。</li>
		<li>将试管放在磁架上5分钟或直到溶液澄清。</li>
		<li>将 50 μL 含有双链 cDNA 的上清液转移到新的 8 条管中。 注意：此时可以停止方案，样品可以储存在-30°C。</li>
	</ol></li>
	<li>cDNA文库的末端制备<ol><li>向洗脱的双链 cDNA 样品中加入 7 μL 末端精纯反应缓冲液（绿色）和 3 μL 末端精纯酶混合物（绿色）。</li>
		<li>以 50 μL 体积设置移液 10 次，在不产生气泡的情况下混合混合物，然后快速旋转样品。</li>
		<li>将样品置于PCR机器中，在20°C孵育30分钟，在65°C孵育30分钟，并在4°C下保持。</li>
	</ol></li>
	<li>接头连接<ol><li>根据制造商的说明稀释适配器（红色）。</li>
		<li>向样品中加入 2.5 μL 稀释的接头、1 μL 连接增强剂（红色）和 30 μL 连接预混液（红色）。</li>
		<li>以 80 μL 体积设置移液 10 次混合混合物，而不会产生气泡，然后快速旋转样品。</li>
		<li>将样品置于PCR机中，并在20°C下孵育15分钟。</li>
		<li>向样品中加入 3 μL 尿嘧啶 DNA 糖基化酶和 DNA 糖基化酶裂解酶核酸内切酶 VIII（蓝色）的混合物并彻底移液。</li>
		<li>将样品放回PCR机中，并在37°C下孵育15分钟，盖子设置为75°C。</li>
	</ol></li>
	<li>纯化连接反应处理的cDNA 注意：使用 SPRIselect 磁珠或可选的 AMpure XP 磁珠的尺寸选择都可用于此目的18.<ol><li>在进行纯化之前预热珠子30分钟，并涡旋以重悬它们。</li>
		<li>将 25 μL 重悬珠转移到步骤 3.6.6 中的 cDNA 样品中，并通过移液至少 10 次彻底混合。</li>
		<li>在室温下孵育5分钟。</li>
		<li>将样品管放在磁性架上5分钟，直到溶液澄清，然后将上清液转移到新的8条管中并丢弃珠子。</li>
		<li>向上清液中加入 10 μL 重悬珠，并通过移液至少 10 次彻底混合。</li>
		<li>将样品管放在磁性架上5分钟，直到溶液澄清，然后弃去上清液，不要干扰珠子。</li>
		<li>将 200 μL 新鲜制备的 80% 乙醇加入带有珠子的试管中，同时将样品保持在磁架上并在室温下孵育 30 秒。</li>
		<li>除去上清液并重复步骤3.7.7一次，但这次小心地除去所有残留的上清液。</li>
		<li>将珠子风干 1-5 分钟，同时将样品保持在磁铁上。 注意： 不要过度干燥珠子;颜色应为深棕色。</li>
		<li>从磁架中取出样品，并通过向磁珠中加入 17 μL 0.1x TE 缓冲液来洗脱 DNA。通过移液至少10次彻底混合，并在室温下孵育2分钟。</li>
		<li>将试管放在磁架上5分钟，直到溶液澄清。</li>
		<li>将含有靶标末端制备的 dsDNA 的 15 μL 上清液转移到新的 8 条管中。</li>
	</ol></li>
	<li>接头连接DNA的PCR富集<ol><li>向接头连接的 DNA 样品中加入 25 μL 预混液（蓝色）、5 μL 5' 接头引物和 5 μL 3' 接头引物。通过移液至少 10 次彻底混合。</li>
		<li>将样品放入PCR仪中，按照以下步骤进行PCR扩增：（1）98°C30秒，（2）98°C10秒，（3）65°C75秒，（4）65°C5分钟，（5）保持在4°C;其中步骤 （2） 和 （3） 应重复 N+2 循环数，其中 N 等于说明书中的 RNA 输入量。这意味着由于在步骤 3.7 中对样品进行双面尺寸选择，因此需要 2 个额外的循环。 注意：例如，建议在 9-10 个循环的 PCR 中使用 8 ng 纯化的 mRNA 起始量;但在步骤 3.7 中使用双侧、大小选择、连接的 cDNA，推荐的 PCR 循环为 11-12 个循环。</li>
	</ol></li>
	<li>PCR扩增文库的纯化<ol><li>在进行纯化之前，将DNA纯化珠预热30分钟，并涡旋以重悬珠。</li>
		<li>将 45 μL （0.9x） 重悬的珠子转移到步骤 3.8.2 中的 PCR 产物中，并通过移液至少 10x 彻底混合。</li>
		<li>在室温下孵育5分钟。</li>
		<li>将样品管放在磁架上5分钟，直到溶液澄清，然后除去上清液并保留含有DNA的珠子。</li>
		<li>将 200 μL 新鲜制备的 80% 乙醇加入到带有珠子的试管中，将样品留在磁架上，并在室温下静置 30 秒。</li>
		<li>取出上清液并重复步骤3.9.5一次，但这次小心地除去所有残留的上清液。</li>
		<li>将珠子风干 1-5 分钟，同时将样品保持在磁铁上。 注意： 不要过度干燥珠子;颜色应为深棕色。</li>
		<li>从磁架上取下样品，向珠子中加入 22 μL 0.1x TE 缓冲液以洗脱 DNA。通过移液至少10次彻底混合，并在室温下孵育2分钟。</li>
		<li>将试管放在磁性架上，静置5分钟或直到溶液在室温下澄清。</li>
		<li>将 20 μL 上清液转移到新的 8 条管中。</li>
		<li>将 2.2 μL 转移到 8 联管中，以评估文库数量和质量。 注：文库的数量也可以通过qPCR检测（参见 材料表）。</li>
		<li>将bsRNA-seq文库样品储存在-20°C。</li>
	</ol></li>
</ol>

创建用于转录组范围作图的稳健亚硫酸氢盐-mRNA 文库

<ol>
	<li>Boccaletto, P., 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=MODOMICS:+a+database+of+RNA+modification+pathways.+2021+update.">MODOMICS: a database of RNA modification pathways. 2021 update.</a> Nucleic Acids Research. 50, D231-D235 (2022).</li><li>Bohnsack, K. E., H&#246;bartner, C., Bohnsack, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+5-methylcytosine+(m5C)+RNA+Methyltransferases:+Mechanisms,+Cellular+Functions,+and+Links+to+Disease.">Eukaryotic 5-methylcytosine (m5C) RNA Methyltransferases: Mechanisms, Cellular Functions, and Links to Disease.</a> Genes. 10 (2), 102 (2019).</li><li>Shinde, H., Dudhate, A., Kadam, U. S., Hong, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+methylation+in+plants:+An+overview.">RNA methylation in plants: An overview.</a> Frontiers in Plant Science. 14, 1132959 (2023).</li><li>Courtney, D. 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=Epitranscriptomic+addition+of+m5C+to+HIV-1+transcripts+regulates+viral+gene+expression.">Epitranscriptomic addition of m5C to HIV-1 transcripts regulates viral gene expression.</a> Cell Host &amp; Microbe. 26 (2), 217-227 (2019).</li><li>Jonkhout, N., 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=The+RNA+modification+landscape+in+human+disease.">The RNA modification landscape in human disease.</a> RNA. 23 (12), 1754-1769 (2017).</li><li>Legrand, 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=Statistically+robust+methylation+calling+for+whole-transcriptome+bisulfite+sequencing+reveals+distinct+methylation+patterns+for+mouse+RNAs.">Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs.</a> Genome Research. 27 (9), 1589-1596 (2017).</li><li>Schaefer, 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+5-methylcytosine+analysis+by+bisulfite+sequencing.">RNA 5-methylcytosine analysis by bisulfite sequencing.</a> Methods in Enzymology. 560, 297-329 (2015).</li><li>Amort, 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=Distinct+5-methylcytosine+profiles+in+poly(A)+RNA+from+mouse+embryonic+stem+cells+and+brain.">Distinct 5-methylcytosine profiles in poly(A) RNA from mouse embryonic stem cells and brain.</a> Genome Biology. 18 (1), 1 (2017).</li><li>Schumann, U., 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=Multiple+links+between+5-methylcytosine+content+of+mRNA+and+translation.">Multiple links between 5-methylcytosine content of mRNA and translation.</a> BMC Biology. 18 (1), 40 (2020).</li><li>Yang, X., 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=5-Methylcytosine+promotes+mRNA+export+-+NSUN2+as+the+methyltransferase+and+ALYREF+as+an+m5C+reader.">5-Methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m5C reader.</a> Cell Research. 27, 606 (2017).</li><li>Chen, X., 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=5-Methylcytosine+promotes+pathogenesis+of+bladder+cancer+through+stabilizing+mRNAs.">5-Methylcytosine promotes pathogenesis of bladder cancer through stabilizing mRNAs.</a> Nature Cell Biology. 21 (8), 978-990 (2019).</li><li>Guo, 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=Disease+activity-associated+alteration+of+mRNA+m(5)+C+methylation+in+CD4(+)+T+cells+of+systemic+lupus+erythematosus.">Disease activity-associated alteration of mRNA m(5) C methylation in CD4(+) T cells of systemic lupus erythematosus.</a> Frontiers in Cell and Developmental Biology. 8 (5), 430 (2020).</li><li>Song, H., 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=Biological+roles+of+RNA+m5C+modification+and+its+implications+in+cancer+immunotherapy.">Biological roles of RNA m5C modification and its implications in cancer immunotherapy.</a> Biomarker Research. 10 (1), 15 (2022).</li><li>Xue, C., Zhao, Y., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+RNA+cytosine-5+methylation:+detection,+regulatory+mechanisms,+biological+functions+and+links+to+cancer.">Advances in RNA cytosine-5 methylation: detection, regulatory mechanisms, biological functions and links to cancer.</a> Biomarker Research. 8 (1), 43 (2020).</li><li>Helm, M., Motorin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+RNA+modifications+in+the+epitranscriptome:+predict+and+validate.">Detecting RNA modifications in the epitranscriptome: predict and validate.</a> Nature Reviews Genetics. 18 (5), 275-291 (2017).</li><li>Guo, 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=Advances+in+mRNA+5-methylcytosine+modifications:+Detection,+effectors,+biological+functions,+and+clinical+relevance.">Advances in mRNA 5-methylcytosine modifications: Detection, effectors, biological functions, and clinical relevance.</a> Molecular Therapy - Nucleic Acids. 26, 575-593 (2021).</li><li>Saplaoura, E., Perrera, V., Colot, V., Kragler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylated+RNA+Immunoprecipitation+Assay+to+Study+m5C+Modification+in+Arabidopsis.">Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis.</a> Journal of Visualized Experiments:JoVE. (159), e61231 (2020).</li><li>Quail, M. A., Swerdlow, H., Turner, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+protocols+for+the+illumina+genome+analyzer+sequencing+system.">Improved protocols for the illumina genome analyzer sequencing system.</a> Current Protocols in Human Genetics. , (2009).</li><li>Chen, S. -. Y., 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=RNA+bisulfite+sequencing+reveals+NSUN2-mediated+suppression+of+epithelial+differentiation+in+pancreatic+cancer.">RNA bisulfite sequencing reveals NSUN2-mediated suppression of epithelial differentiation in pancreatic cancer.</a> Oncogene. 41 (22), 3162-3176 (2022).</li><li>Squires, J. E., 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=Widespread+occurrence+of+5-methylcytosine+in+human+coding+and+non-coding+RNA.">Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA.</a> Nucleic Acids Research. 40 (11), 5023-5033 (2012).</li><li>Han, X., 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=Dynamic+DNA+5-hydroxylmethylcytosine+and+RNA+5-methycytosine+reprogramming+during+early+human+development.">Dynamic DNA 5-hydroxylmethylcytosine and RNA 5-methycytosine reprogramming during early human development.</a> Genomics, Proteomics &amp; Bioinformatics. , (2022).</li><li>Amort, 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=Long+non-coding+RNAs+as+targets+for+cytosine+methylation.">Long non-coding RNAs as targets for cytosine methylation.</a> RNA biology. 10 (6), 1003-1008 (2013).</li><li>Sun, Z., 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=Effects+of+NSUN2+deficiency+on+the+mRNA+5-methylcytosine+modification+and+gene+expression+profile+in+HEK293+cells.">Effects of NSUN2 deficiency on the mRNA 5-methylcytosine modification and gene expression profile in HEK293 cells.</a> Epigenomics. 11 (4), 439-453 (2019).</li><li>Huang, T., Chen, W., Liu, J., Gu, N., Zhang, R. <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+identification+of+mRNA+5-methylcytosine+in+mammals.">Genome-wide identification of mRNA 5-methylcytosine in mammals.</a> Nature Structural and Molecular Biology. 26 (5), 380-388 (2019).</li><li>Rieder, D., Amort, T., Kugler, E., Lusser, A., Trajanoski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=meRanTK:+methylated+RNA+analysis+ToolKit.">meRanTK: methylated RNA analysis ToolKit.</a> Bioinformatics. 32 (5), 782-785 (2015).</li><li>Kraus, A. J., Brink, B. G., Siegel, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+specific+oligo-based+depletion+of+rRNA.">Efficient and specific oligo-based depletion of rRNA.</a> Scientific Reports. 9 (1), 12281 (2019).</li><li>Edelheit, S., Schwartz, S., Mumbach, M. R., Wurtzel, O., Sorek, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome-wide+mapping+of+5-methylcytidine+RNA+modifications+in+bacteria,+archaea,+and+yeast+reveals+m(5)C+within+archaeal+mRNAs.">Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m(5)C within archaeal mRNAs.</a> PLoS Genetics. 9 (5), e1003602 (2013).</li><li>Furlan, M., 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=Computational+methods+for+RNA+modification+detection+from+nanopore+direct+RNA+sequencing+data.">Computational methods for RNA modification detection from nanopore direct RNA sequencing data.</a> RNA Biology. 18, 31-40 (2021).</li><li>Leger, 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=RNA+modifications+detection+by+comparative+Nanopore+direct+RNA+sequencing.">RNA modifications detection by comparative Nanopore direct RNA sequencing.</a> Nature Communications. 12 (1), 7198 (2021).</li><li>Mateos, P. 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=Identification+of+m6A+and+m5C+RNA+modifications+at+single-molecule+resolution+from+Nanopore+sequencing.">Identification of m6A and m5C RNA modifications at single-molecule resolution from Nanopore sequencing.</a> bioRxiv. , (2022).</li><li>Johnson, Z., Xu, X., Pacholec, C., Xie, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evaluation+of+parameters+in+RNA+bisulfite+sequencing+data+generation+and+analysis.">Systematic evaluation of parameters in RNA bisulfite sequencing data generation and analysis.</a> NAR Genomics and Bioinformatics. 4 (2), 045 (2022).</li><li>Zhang, Z., 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=Systematic+calibration+of+epitranscriptomic+maps+using+a+synthetic+modification-free+RNA+library.">Systematic calibration of epitranscriptomic maps using a synthetic modification-free RNA library.</a> Nature Methods. 18 (10), 1213-1222 (2021).</li><li>Chao, H. -. P., 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=Systematic+evaluation+of+RNA-Seq+preparation+protocol+performance.">Systematic evaluation of RNA-Seq preparation protocol performance.</a> BMC Genomics. 20 (1), 571 (2019).</li></ol>

作者没有要披露的利益冲突。

在该协议中，通过使用标准化组件实现了聚（A）富集，亚硫酸氢盐转化和文库制备的详细流程。进一步的测序分析提供了目标样品中mRNA 5-甲基胞嘧啶的鉴定。
关键步骤是起始材料（总RNA）的质量，因为RNA的降解会影响poly（A） RNA纯化的回收率。在进行poly（A） RNA纯化步骤之前，应小心处理样品并避免RNase污染。该过程的另一个关键部分是文库制备中的PCR循环次数。循环数的?...

在 150 多种转录后修饰1 中，已在各种类型的 RNA 中鉴定出 5-甲基胞嘧啶 （m5C） 修饰，包括核糖体 RNA、转移 RNA、信使 RNA、micro RNA、长链非编码 RNA、vault RNA、增强子 RNA 和小 cajal 体特异性 RNA2。RNA m 5 C 与多种生物学和病理机制有关，例如调节植物根系发育3、病毒基因表达4 和癌症进展5。该协议的目的是提供简化的管道，以表征处于不同发育阶段或疾病环境中的生物样品的转录组范围的mRNA m5C修饰谱。开发了全转录组亚硫酸氢盐测序来表征亚硫酸氢盐转化 RNA 中碱基对分辨率为 6、7、8、9 的位置和定量胞嘧啶甲基化水平。这在研究 m5C 与参与细胞生物调控机制的基因表达和 RNA 命运的关联时特别有用。在哺乳动物细胞中，有两种已知的 m 5 C 读取器：ALYREF 可以识别细胞核的 m 5 C 并充当 mRNA 细胞核到胞质溶胶转运蛋白10，而 YBX1 可以识别细胞质中的 m5C 并增加 mRNA 稳定性11。系统性红斑狼疮 CD4+ T 细胞中报告了与免疫通路相关的异常 m5C mRNA12。研究表明，mRNA m5C 修饰与癌症免疫调节和癌症进展之间存在关联13,14。因此，在mRNA上绘制m5C修饰图谱可以为阐明潜在的调控机制提供关键信息。
为了研究RNA m 5 C修饰在某些生物学条件下的功能作用，基于亚硫酸氢盐转化（bsRNA-seq）和基于抗体亲和富集的方法，如m 5 C-RIP-seq、miCLIP-seq和5-Aza-seq，可以与高通量测序平台相结合，在转录组范围内使用m5C修饰对靶区和序列进行高效检测15，16.该协议的优势在于，由于基于抗体亲和富集的方法依赖于高质量抗体的可用性，因此该协议的优势在于单碱基分辨率下提供了全面的RNA m 5 C景观，并且可以实现m5C甲基化景观的单片段分辨率17。
所有 RNA 样品都将使用 oligo（dT） 微球进行两轮 mRNA 富集、三个亚硫酸氢盐反应循环和测序文库制备。为了监测 RNA 质量，将在 mRNA 纯化和亚硫酸氢盐反应程序前后通过毛细管凝胶电泳检查每个 RNA 样品，以评估片段分布。纯化的文库将通过其 PCR 扩增子质量进行检查，通过毛细管凝胶电泳检查 DNA 大小分布片段，并在测序前通过基于荧光染料的定量测定检查其总量。该系统还可用于分析广谱生物样品，如农产品、分离的病毒粒子、细胞系、模式生物和病理标本。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agilent 2100 Electrophoresis Bioanalyzer System</td>
			<td>Agilent, Santa Clara, CA</td>
			<td></td>
			<td>RNA quality detection</td>
		</tr>
		<tr>
			<td>AMpure XP beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>purify DNA</td>
		</tr>
		<tr>
			<td>Bioanalyzer DNA high sensitivity kit</td>
			<td>Agilent, Santa Clara, CA</td>
			<td>5067-4626</td>
			<td>DNA quality dection</td>
		</tr>
		<tr>
			<td>Bioanalyzer RNA 6000 Pico kit</td>
			<td>Agilent, Santa Clara, CA</td>
			<td>5067-1513</td>
			<td>RNA quality dection</td>
		</tr>
		<tr>
			<td>DiaMag02 - magnetic rack</td>
			<td>Diagenode, Denville, NJ</td>
			<td>B04000001</td>
			<td>assist library preparation</td>
		</tr>
		<tr>
			<td>DiaMag1.5 - magnetic rack</td>
			<td>Diagenode, Denville, NJ</td>
			<td>B04000003</td>
			<td>assist poly(A) RNA purificaion</td>
		</tr>
		<tr>
			<td>Dynabeads mRNA DIRECT purification kit</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>61011</td>
			<td>poly(A) RNA purificaion; Wash Buffer 1 and Wash Buffer 2</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>J.T.Baker</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ RNA methylation kit</td>
			<td>Zymo, Irvine, CA</td>
			<td>R5002</td>
			<td>bisulfite treatment</td>
		</tr>
		<tr>
			<td>Firefly luciferase mRNA</td>
			<td>Promega, Madison, WI, USA</td>
			<td>L4561</td>
			<td>spike in control seqeunce&#160;</td>
		</tr>
		<tr>
			<td>KAPA Library Quantification Kits</td>
			<td>Roche, Switzerland</td>
			<td>KK4824</td>
			<td>library quantification</td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotometer</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td></td>
			<td>Total RNA quantity detection</td>
		</tr>
		<tr>
			<td>NEBNext multiplex Oligos for illumina (index Primer set1)</td>
			<td>New England Biolabs, Ipswich, MA</td>
			<td>E7335S</td>
			<td>library preparation</td>
		</tr>
		<tr>
			<td>NEBNext Ultra <img alt="Equation 1" src="/files/ftp_upload/65352/65352eq01.jpg" style="margin: auto;" /> Directional RNA Library Prep Kit for Illumina</td>
			<td>New England Biolabs, Ipswich, MA</td>
			<td>E7760S</td>
			<td>library preparation</td>
		</tr>
		<tr>
			<td>Nuclease-free Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>P2 pipetman</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>4641010</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 fluorometer&#160;</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td></td>
			<td>RNA quantity detection</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>Q32854</td>
			<td>DNA quantity detection</td>
		</tr>
		<tr>
			<td>Qubit RNA HS Assay Kit</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>Q32852</td>
			<td>RNA quantity detection</td>
		</tr>
	</tbody>
</table>

enrichment of mrna and bisulfite-mrna library preparation for next-generation sequencing

各种类型的 RNA 转录本中的 RNA 转录后修饰与真核细胞中的多种 RNA 调控有关。异常的 RNA 5-甲基胞嘧啶修饰和 RNA 甲基转移酶的失调表达已被证明与包括癌症在内的多种疾病有关。开发了全转录组亚硫酸氢盐测序，以表征亚硫酸氢盐转化的 RNA 在碱基对分辨率下的位置和定量胞嘧啶甲基化水平。在此，该协议详细介绍了两轮poly（A）RNA纯化，三个亚硫酸氢盐反应循环和文库制备的程序，以允许mRNA 5-甲基胞嘧啶修饰位点的转录组范围定位。主反应后RNA数量和质量的评估对于监测RNA完整性至关重要，也是确保高质量测序文库的关键步骤。理想情况下，这些程序可以在三天内完成。通过该方案，使用高质量的总RNA作为输入，实际上可以建立强大的亚硫酸氢盐-mRNA文库，用于从目标样品进行二代测序。

Among over 150 types of post-transcriptional modifications1, 5-methylcytosine (m5C) modification has been identified in various types of RNAs, including ribosomal RNA, transfer RNA, messenger RNA, micro RNA, long non-coding RNA, vault RNA, enhancer RNA, and small cajal body-specific RNAs2. The RNA m5C is associated with diverse biological and pathological mechanisms such as regulating plant root development3, viral gene expression4, and cancer progression5. The aim of this protocol is to provide streamlined pipelines to characterize the transcriptome-wide mRNA m5C modification profile of biological samples in different developmental stages or in the disease setting. Transcriptome-wide bisulfite-sequencing was developed to characterize the positions and the quantitative cytosine methylation levels in the bisulfite-converted RNA at base-pair resolution6,7,8,9. This is particularly useful when studying the association of m5C with gene expression and RNA fate that is involved in the biological regulatory mechanisms in cells. In the mammalian cell, there are two known m5C readers: ALYREF can recognize m5C at the nucleus and serves as an mRNA nucleus-to-cytosol transporter10, while YBX1 can recognize m5C in the cytoplasm and increase mRNA stabilization11. Aberrant m5C mRNAs related to immune pathways were reported in Systemic Lupus Erythematosus CD4+ T cells12. Studies have revealed an association between mRNA m5C modification and modulation of cancer immunity and cancer progression13,14. Hence, mapping the m5C modification profile on the mRNA can provide crucial information to elucidate the potential regulatory machinery.
To investigate the functional roles of RNA m5C modification under certain biological conditions, the bisulfite conversion-based (bsRNA-seq) and antibody affinity enrichment-based approaches such as m5C-RIP-seq, miCLIP-seq, and 5-Aza-seq can be combined with the high-throughput sequencing platform to provide efficient detection of targeted regions and sequences with the m5C modifications on a transcriptome-wide scale15,16. The advantage of this protocol provides the comprehensive RNA m5C landscape at a single-base resolution since the antibody affinity enrichment-based approach relies upon the availability of high-quality antibodies and could achieve the single-fragment resolution of m5C methylation landscape17.
All RNA samples will be processed with two rounds of mRNA enrichment using oligo(dT) beads, three cycles of bisulfite reaction, and the sequencing library preparation. To monitor the RNA quality, each RNA sample will be examined by capillary gel electrophoresis before and after the procedures of mRNA purification and bisulfite reaction to assess fragment distribution. The purified libraries will be examined by their PCR amplicon qualities, DNA size distribution fragments by capillary gel electrophoresis, and their overall quantities examined by fluorescence dyes-based quantitative assays before sequencing. The system can also be used to analyze a broad spectrum of biological samples such as agricultural produce, isolated virions, cell lines, model organisms, and pathological specimens.

作者聚焦：解码 RNA 甲基化在胰腺癌中的作用 - 一项单碱基分辨率研究 

mRNA和亚硫酸氢盐-mRNA文库的富集制备用于二代测序

This research aims to study the role of RNA methylation, specifically 5-methylcytosine modification, in pancreatic cancer. The questions we want to answer are how upregulation or downregulation of certain RNA methyltransferase affects cancer cell phenotypes and also, through which RNA substrates. This study provides the comprehensive RNA m5C landscape at a single-base resolution with standardized equipment.From this study using bisulfite RNA seq, the results links the association of RNA m5C modification with poor differentiation of cancer cells. This should pave the way for future studies in discerning passenger versus disease driver effects of specific m5C in the RNA transcripts. With this technique, we can identify the set of differentially-methylated RNAs that may participate in tumor progression.Further, single molecule base experiments can provide a more detailed regulatory role of specific RNA m5C.

按照本报告中的程序从细胞系19 中生成一系列bsRNA-seq文库 （图1）。在对细胞系样品进行总RNA纯化并进行DNase处理并通过凝胶电泳和紫外-可见分光光度法（A260/A280）检查质量后，RNA样品可以进行poly（A）RNA富集。为了确定双重纯化是否可以去除大部分核糖体RNA，通过毛细管电泳总RNA测定法评估poly（A） RNA的纯化效率，该测定法可以自动计算rRNA污...

Watch this Scientific Journal Video about Decoding RNA Methylation's Role in Pancreatic Cancer - A Single-Base Resolution Study at JoVE.com

该协议提供了一种易于遵循的工作流程，可使用标准化设备对感兴趣的生物样品进行poly（A）RNA纯化、亚硫酸氢盐转化和文库制备。

RNA post-transcriptional modifications in various types of RNA transcripts are associated with diverse RNA regulation in eukaryotic cells. Aberrant RNA ...

本研究旨在研究 RNA 甲基化，特别是 5-甲基胞嘧啶修饰在胰腺癌中的作用。我们想要回答的问题是某些RNA甲基转移酶的上调或下调如何影响癌细胞表型，以及RNA底物通过哪些表型。本研究使用标准化设备以单碱基分辨率提供全面的 RNA m5C 图景。从这项使用亚硫酸氢盐RNA seq的研究中，结果将RNA m5C修饰与癌细胞分化不良的关联联系起来。这应该为未来的研究铺平道路，以辨别RNA转录本中特定m5C的乘客与疾病驱动因素的影响。通过这种技术，我们可以识别可能参与肿瘤进展的一组差异甲基化RNA。此外，单分子碱基实验可以提供特异性RNA m5C的更详细的调控作用。

enrichment-mrna-bisulfite-mrna-library-preparation-for-next

Research

JoVE Journal

Biology

Enrichment of mRNA and Bisulfite-mRNA Library Preparation for Next-Generation Sequencing

828 次观看.  National Cheng Kung University. 本研究旨在研究 RNA 甲基化，特别是 5-甲基胞嘧啶修饰在胰腺癌中的作用。我们想要回答的问题是某些RNA甲基转移酶的上调或下调如何影响癌细胞表型，以及RNA底物通过哪些表型。本研究使用标准化设备以单碱基分辨率提供全面的 RNA m5C 图景。从这项使用亚硫酸氢盐RNA seq的研究中，结果将RNA m5C修饰与癌细胞分化不良的关联联系起来。这应该为未来的研究铺平道路，以辨...

视频: 作者聚焦：解码 RNA 甲基化在胰腺癌中的作用 - 一项单碱基分辨率研究 

RNA post-transcriptional modifications in various types of RNA transcripts are associated with diverse RNA regulation in eukaryotic cells. Aberrant RNA 5-methylcytosine modifications and the dysregulated expression of RNA methyltransferases have been shown to be associated with various diseases, including cancers. Transcriptome-wide bisulfite-sequencing was developed to characterize the positions and the quantitative cytosine methylation levels in the bisulfite-converted RNA at the base-pair resolution. Herein, this protocol presents the procedures of two rounds of poly(A) RNA purification, three cycles of bisulfite reaction, and library preparation in detail to allow the transcriptome-wide mapping of mRNA 5-methylcytosine modification sites. The assessment of RNA quantity and quality after the main reaction is essential to monitor RNA integrity and is a critical step for ensuring high-quality sequencing libraries. Ideally, the procedures can be completed within three days. With this protocol, using high-quality total RNA as the input can practically build up robust bisulfite-mRNA libraries for next-generation sequencing from the sample of interest.

This protocol provides an easy-to-follow workflow to conduct poly(A) RNA purification, bisulfite conversion, and library preparation using standardized equipment for a biological sample of interest.

科研

生物学

Poly(A) RNA Purification and Bisulfite Conversion for Next Generation Sequencing

To begin, prepare 10 to 20 micrograms of high-quality total RNA for the isolation of poly(A)enriched RNA. Transfer an aliquot of RNA into a nuclease free 1.5 milliliter microcentrifuge tube. Incubate the tube in a heat block at 70 degrees Celsius for five minutes, and then place it on ice for two minutes.After resuspending the oligo(dT)beads, transfer the required volume of bead suspension to a new 1.5 milliliter microcentrifuge tube and place it on the magnet for three minutes until the magnetic beads form a pellet. Remove the supernatant and add an equal volume of lysis buffer. Resuspend the beads.Place the tube back on the magnet for three minutes and remove the supernatant. For the first round of purification, add lysis buffer to the RNA sample tube and mix thoroughly. Then transfer the mixture to the oligo(dT)beads and resuspend fully pipetting at least 10 times.Allow the samples to incubate with continuous rotation at room temperature. After 15 minutes, place the sample on a magnet for five minutes or until the supernatant is clear. Discard the supernatant.Add 600 microliters of wash buffer one to the beads and carefully mix to resuspend. Next, add 300 microliters of wash buffer two to the beads and carefully mix to resuspend. Place the tube on the magnet for five minutes or until the supernatant is clear.Discard the supernatant. Next, add 30 microliters of cold 10 millimolar tris HCL to the sample tube and incubate at 70 degrees Celsius. After five minutes, quickly transfer the tube on the magnet.And when the suspension is clear, transfer the supernatant containing the eluted poly(A)enriched RNA into a new 1.5 milliliter microcentrifuge tube. For the second round of purification, add 120 microliters of lysis buffer to the eluted RNA sample and mix thoroughly. Wash the beads used in the first round of purification with an equal volume of lysis buffer.Pipette 10 times to resuspend the beads and then place the tube on the magnet for five minutes before discarding the supernatant. Transfer the RNA lysis buffer mixture to the washed beads and mix thoroughly by pipetting. After washing the beads as demonstrated previously, add 25 microliters of cold 10 millimolar tris HCL to the sample tube and incubate at 70 degrees Celsius.After five minutes, quickly transfer the tube to the magnet, and when the suspension is clear, transfer the supernatant containing eluted poly(a)enriched RNA to a new 1.5 milliliter microcentrifuge tube. Next, for bisulfite conversion, transfer 19 microliters of purified poly(A)enriched RNA sample to a 0.2 milliliter eight strip tube and add one microliter of spiking control at the predetermined one to 10, 000 quantity ratio. After adding 130 microliters of conversion reagent to the tubes and thoroughly mixing, place the tube in the PCR machine and start the PCR program.After PCR, place the column onto the collection tube and add 250 microliters of RNA binding buffer to the column. Then transfer 150 microliters of bisulfite treated sample to the column and pipette thoroughly. Add 400 microliters of 95 to 100%ethanol to the column, quickly close the cap and invert several times.After repeated centrifugation, washing, and neutralization with desulphonation buffer, transfer the column to a new 1.5 milliliter microcentrifuge tube. Add 20 to 30 microliters of nuclease-free water to the column and allow it to stand for one minute. Centrifuge at 10, 000 G for 30 seconds at 25 degrees Celsius and transfer 2.2 microliters of supernatant to eight strip tubes to assess RNA quantity and quality.Poly(A)RNA enrichment efficiency was assessed by capillary electrophoresis total RNA assay, resulting in decreased ribosomal RNA contamination after double purification in pancreatic cancer cell lines. RNA quantity before and after bisulfite treatment was assessed by capillary electrophoresis. After the bisulfite treatment, the RNA size distribution showed a 200 to 500 nucleotide peak because of fragmentation caused by the bisulfite reaction.Capillary electrophoresis of the amplicon showed successful library preparation with minimal primer remaining and no over amplification peak. After the sequencing reads aligned to the reference sequence, the average of 2, 440 of total reads were mapped to the spiking sequence, and the total analyzed C to T conversion rate reached an average of 99.81%