{{'Search' | translate}}
 

AMPureXP beads

Agencourt AMPure XP - PCR纯化

Company: Beckman Coulter
Catalog#: A63881
Bio-protocol()
Company-protocol()
Other protocol()

Isolation of Nuclei in Tagged Cell Types (INTACT), RNA Extraction and Ribosomal RNA Degradation to Prepare Material for RNA-Seq
Author:
Date:
2018-04-05
[Abstract]  Gene expression is dynamically regulated on many levels, including chromatin accessibility and transcription. In order to study these nuclear regulatory events, we describe our method to purify nuclei with Isolation of Nuclei in TAgged Cell Types (INTACT). As nuclear RNA is low in polyadenylated transcripts and conventional pulldown methods would not capture non-polyadenylated pre-mRNA, we also present our method to remove ribosomal RNA from the total nuclear RNA in preparation for nuclear RNA-Seq. [摘要]  基因表达在多个水平上动态调节,包括染色质可及性和转录。 为了研究这些核调节事件,我们描述了我们用净化细胞核(INTACT)纯化细胞核的方法。 由于核RNA在聚腺苷酸化转录物中低并且常规下拉方法不会捕获非聚腺苷酸化前mRNA,所以我们还提出了我们的方法以从核RNA总RNA中去除核糖体RNA以准备核RNA-Seq。

【背景】分离用于基因表达实验的特定细胞类型降低了噪音并提高了实验的精确度和发现的不同表达基因的数量。用于细胞类型特异性研究的各种方法被广泛使用,每种方法都有其优点和缺点(Bailey-Serres,2013年综述)。分离特定的调控区室,如细胞核(来自细胞器,核糖体,细胞质,等等)可以进一步精确解析调控基因表达的分子事件。在这里,我们描述了一种方法,可以从冷冻组织中分离特定细胞类型的细胞核,适用于研究核基因表达的实验(例如,核RNA的RNA-Seq,ATAC-Seq,ChIP -Seq,等。)。此外,我们描述了RNA的处理,导致材料适合作为RNA-Seq实验的输入。这里描述的方案与水稻根组织(日本野生稻栽培品种日本晴)(Reynoso等人,2018年)一起使用,但是它们基于以前开发的方案, (拟订和Henikoff,2010年和2011年)和番茄(罗恩等人,2014年)。

该协议的第一部分,INTACT方法(用于标记特定细胞类型的核的分离)允许体内亲和标记和随后从感兴趣的细胞类型中纯化细胞核。这是通过由包膜靶向结构域,GFP和生物素连接酶识别肽(BLRP)组成的三联核标签融合蛋白(NTF)的细胞类型特异性表达实现的。 ...

Reduced Representation Bisulfite Sequencing in Maize
Author:
Date:
2018-03-20
[Abstract]  DNA methylation is an epigenetic modification that regulates plant development (Law and Jacobsen, 2010). Whole genome bisulfite sequencing (WGBS) is a state-of-the-art method for profiling genome-wide methylation patterns with single-base resolution (Cokus et al., 2008). However, for an organism with a large genome, e.g., the 2.1 Gb genome of maize, WGBS may be very expensive. Reduced representation bisulfite sequencing (RRBS) has been developed in mammalian studies (Smith et al., 2009). By digesting the genome with MspI with a size selection range of approximately 40-220 bp, CG-rich regions covering only ~1% of the human genome can be specifically sequenced. However, unlike mammalian genomes, plant genomes do not exhibit clear CpG islands. Therefore ... [摘要]  DNA甲基化是调节植物发育的表观遗传修饰(Law and Jacobsen,2010)。全基因组亚硫酸氢盐测序(WGBS)是用单碱基分辨率分析全基因组甲基化模式的最先进的方法(Cokus et al。,2008)。然而,对于具有大基因组的生物体,例如玉米的2.1Gb基因组,WGBS可能非常昂贵。代表性亚硫酸氢盐测序(RRBS)已经在哺乳动物研究中发展(Smith等人,2009)。通过用大小选择范围大约40-220bp的 Msp 消化基因组,可以对仅涵盖〜1%人类基因组的CG富含区域进行特异性测序。然而,与哺乳动物基因组不同,植物基因组不显示清楚的CpG岛。因此原来的RRBS协议不适用于工厂。因此,我们开发了一种计算机管道来选择特定的酶以生成感兴趣区域(ROI) - 富集的,例如,富含启动子的,减少的植物表达基因组(例如, Hsu et al。,2017)。通过用MseI消化玉米基因组并选择40-300bp片段,我们测序了大约四分之一的玉米基因组,同时保留了84.3%的启动子信息。该协议已在玉米中成功建立,可广泛应用于任何基因组。我们的计算机管道系统与RRBS文库制备方案相结合,允许进行计算分析和实验验证。

【背景】DNA甲基化是一种可遗传的表观遗传修饰,通过调节基因表达和染色质结构在动物,植物和真菌的许多发育过程中发挥重要作用(Law and ...

Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq
Author:
Date:
2018-02-20
[Abstract]  Most nematodes are small worms that lack enough RNA for regular RNA-seq protocols without pooling hundred to thousand of individuals. We have adapted the Smart-seq2 protocol in order to sequence the transcriptome of an individual worm. While developed for individual Steinernema carpocapsae and Caenorhabditis elegans larvae as well as embryos, the protocol should be adaptable for other nematode species and small invertebrates. In addition, we describe how to analyze the RNA-seq results using the Galaxy online environment. We expect that this method will be useful for the studying gene expression variances of individual nematodes in wild type and mutant backgrounds. [摘要]  大多数线虫是小蠕虫,缺乏足够的RNA用于常规的RNA-seq协议,而没有汇集成千上万的个体。 我们已经调整了Smart-seq2协议来排序单个蠕虫的转录组。 虽然针对Steinernema carpocapsae和Caenorhabditis elegans幼虫以及胚胎开发,但该方案应该适用于其他线虫物种和小无脊椎动物。 另外,我们介绍如何使用Galaxy在线环境分析RNA-seq结果。 我们预计这种方法将有助于研究野生型和突变体背景个体线虫的基因表达差异。

【背景】低输入RNA-seq方案和扩增试剂盒,例如Smart-seq(Takara Bio,USA,Inc)和SuperAmp(Miltenyl Biotec,Inc),已经越来越多地开发和商业化,作为对低输入RNA-基于小组织,单一微生物和单细胞的seq研究。这些研究经常探索并解决特定群体(例如细胞群体,复杂组织或微生物群体)的个体中的异源基因表达。针对微生物(如线虫)的低输入RNA-seq方案的改进和适应将通过允许在单一线虫水平上分析基因表达异质性而极大地有益于线虫领域。在这里,我们已经调整了单细胞RNA-seq方案Smart-seq2(Picelli等人,2013和2014; Trombetta等人,2014),对于单线虫RNA测序。我们成功地在昆虫寄生线虫Steinernema ...

Comments