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Illumina/TruSeq Ribo Profile for Mammalian (12 reactions, 12 indexes)/RPHMR12126/1 Ea

Illumina/TruSeq Ribo Profile for Mammalian (12 reactions, 12 indexes)/RPHMR12126/1 Ea
  • Illumina/TruSeq Ribo Profile for Mammalian (12 reactions, 12 indexes)/RPHMR12126/1 Ea
商品介绍

ProductHighlights:

  • StudyactivemRNAtranslationwithsequencing
  • Sequenceribosome-protectedmRNA
  • Predictproteinabundance
  • Investigatetranslationalcontrol
  • Measuregeneexpression
  • Rapid,scalablespin-columnmethod
  • Noultracentrifugerequired
  • CompatIBLewithyeastandmammaliansamples
Sequenceactivelytranslatedtranscripts

Ribosomeprofilingisatechniqueforinvestigatingtranslationalcontrolthatprovidesasnapshotofalltheribosomesactiveinacellataspecifictime.TruSeqRiboProfilegeneratesRNA-Seqlibrariesfromribosome-protectedfragmentsofmRNAsthatareactivelybeingtranslated.

UseTruSeqRiboProfiletosequencemRNAfragmentsundergoingtranslationbyribosomes.ThesemRNAfragmentsarecalled"footprinted"orribosome-protectedmRNAfragments.

YoucanalsoidentifyproteinsbeingactivelytranslatedfromsamplespreparedwithTruSeqRiboProfile.Samplescollectedatdifferenttimesoftenshowchangesintranslation.Samplestreatedwithdifferentdrugsoftenshowdifferenttranslationpatterns.

Specifications:

InputQuantity0.1–1ughigh-qualitypurifiedtotalRNAfromblood
ContentSpecificationsCapturescodingRNAplusmultipleformsofnon-codingRNA
MechanismofActionBead-basedrRNAdepletion,CDNAsynthesis,andPCR
MultiplexingLow-throughputkits:Poolupto12samples.Orpoolupto24sampleswithsetsAandBtogether,High-throughputkitversion:Prepare96uniquelyindexedsamples
SystemCompatibilityNovaSeq5000,GenomeAnalyzerIIx,HiSeq2000,NextSeq500,HiSeq2500
AutomationCapABIlityLiquidHandlingRobots
VariantClassTranscriptVariants
SpeciesCategoryRat
品牌介绍

Illumina公司,致力于新一代测序和芯片技术的生产与开发,提供最新的产品与应用资讯。Illumina公司创立于1998年4月,是遗传变异和生物学功能分析领域的优秀的产品、技术和服务供应商。通过帮助客户加快实现生物信息的采集、分析和应用,来改善人类健康。

As a startup, Illumina aspired to transform human health. Our initial products enabled researchers to explore DNA at an entirely new scale, helping them create the first map of gene variations associated with health, disease, and drug response. Every breakthrough opened up a new world, and showed us how much further there is to go

While the rate of progress is accelerating exponentially, we are only beginning to understand the clinical significance of the genome. What causes a cancer cell to mutate? What is the origin of a puzzling disease? Is it possible to prevent the next outbreak? Or safeguard the world’s food supply? These are just a few of the challenges that inspire us to push the boundaries of our imagination

Today we are a global leader in genomics – an industry at the intersection of biology and technology. At the most fundamental level, we enable our customers to read and understand genetic variations. We strive to make our solutions increasingly simple, more accessible, and always reliable. As a result, discoveries that were unimaginable even a few years ago are now becoming routine – and are making their way into patient treatment.

We now have the ability to sequence at an unprecedented scale. Collectively, this will give us a much deeper understanding of genetics than ever before. We will begin to truly unlock the power of the genome. These advances will trigger a fundamental shift in healthcare and beyond. Medicine will continue to become more preventive and more precise. We will be healthier, longer. We have only just begun.

At Illumina, our goal is to apply innovative technologies to the analysis of genetic variation and function, making studies possible that were not even imaginable just a few years ago. It is mission critical for us to deliver innovative, flexible, and scalable solutions to meet the needs of our customers. As a global company that places high value on collaborative interactions, rapid delivery of solutions, and providing the highest level of quality, we strive to meet this challenge. Illumina innovative sequencing and array technologies are fueling groundbreaking advancements in life science research, translational and consumer genomics, and molecular diagnostics



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    Illumina测序流程简介


    二代测序也称大规模平行测序。


    主要平台有3家:Illumina的HiSeq、MiSeq以及早期的GA;Roche的454;Life Technologies (即ABI,现ThermoFisher)的SOLiD、Ion Torrent和Ion Proton如今Roche已经停产。


    简单归纳,3家的原理如下:

    Roche 454 FLX:Pyrosequencing(焦磷酸测序),检测焦磷酸转换成的荧光信号,emulsion PCR;

    Illumina: Sequencing-by-synthesis (边合成边测序)with reversible terminators, bridge PCR;

    ABI SOLiD: Sequencing by ligation(边连接边测序), emulsion PCR;

    Thermo Ion Torrent: Pyrosequencing(焦磷酸测序), 检测质子电位(pH值),emulsion PCR。



    Illumina技术是所有二代测序技术路线中最简便的,直奔目的,不绕弯子。因为简单直接,所以稳定可靠,重复性强,数据质量高,错误率低。


    illumina整个测序分4步:

    核酸提取,文库制备,簇生成,测序,数据分析。

    上机前:核酸提取,文库制备,簇生成。

    上机后:测序及数据分析。


    A核酸提取

    样本类型:培养的细胞、外周血、新鲜冷冻组织或石蜡包埋组织(FFPE)

    提取核酸类型:DNA、总RNA或者小RNA


    步骤:

    样本提取-纯化-定量

    标准流程需求:0.1-1ug DNA或RNA

    试剂:提取及纯化试剂盒

    定量仪器: NanoDrop 显示A260/280,可显示纯度

    1标准流程2 低量流程(依自条件和经验而定)

    B 文库构建

    文库制备的具体流程因样本种类和测序目的的不同而不同,比如基因组DNA,转录组(mRNA),microRNA, CHiP-Seq,外显子捕获测序等等,但是大同小异,基本过程是把长链DNA随机打断成短片段,两端接上通用引物,再经过12循环左右的PCR把文库放大。

    基因组文库构建步骤:

    基因组DNA--350bp左右DN**段—barcode接头序列—12循环左右文库扩增片段化、连接、扩增“三板斧” 期间穿插进行一些纯化、定量的辅助步骤。

    1 片段化:

    原则是断点越随机越好,片段长度越集中越好

    方法:高压气体的雾化作用、超声波的气穴作用、普通超声波、酶等手段、设备。


    一般而言:

    SR测序的模板长度约350碱基,PE测序的模板长度约500碱基,MP测序的模板长度约1k-10k碱基

    超声方法片段化后片段大小呈正态分布,通过琼脂糖电泳割胶或者用不同缓冲液下磁珠纯化方法来进一步收缩片段长度范围,精选所需片段。

    2末端修补

    一般而言,末端修补要用到3种酶:5’端延伸的酶,5’端连接的酶,以及3’端延伸的酶。

    3 3’端加A

    目的:为了提高连接效率,也为了防止接头与插入片段以多种方向连接,同时减少接头之间的互联,接头与插入片段之间的连接采用TA半粘性末端的连接方式。

    方法:接头的3’端带有一个突出的T碱基,插入片段的3’端带有一个突出的A碱基。

    4 两端加接头


    目的:厂家提供的接头都包含有序列已知的barcode区域,以方便测序完成后对数据进行拆分。这些接头按barcode序列进行编号,所以在进行接头连接这一步实验操作的时候,要特别小心不要把barcode编号与样本之间的对应关系搞错。

    另外,不同的应用,其接头在序列和结构上都有可能不一样。比如SR测序与PE测序,它们的接头和flow cell都不一样,不能混用。

    5 连接产物纯化

    目的:选出完整的连接产物,去除残缺不全的副产物

    方法:割胶回收;磁珠纯化

    6 PCR富集

    目的:增加文库量,方便上级测序;提高完整片段文库所占比例

    注意:此步可省略,因为PCR会引入数据偏倚率,若不喜欢,可省略,称为PCR-free

    7 过柱 磁珠纯化

    8 文库质检

    lAgilent 2100 Bioanalyzer:测定文库片段长度及长度分布;

    lQubit或其他设备:测定浓度;

    l琼脂糖凝胶电泳;

    Qubit只取其测定的重量浓度(ng/uL);2100只取其测定的文库片段长度。二者结合,计算文库的摩尔浓度;琼脂糖凝胶电泳用于观察文库的整体情况,片段长度是否符合预期,片段长度分布情况,是否有杂带,是否有引物二聚体或接头二聚体残留等等。如果有二聚体残留,则进行纯化。

    结合三者数据,计算文库摩尔浓度,将文库统一稀释到平台要求的浓度

    根据项目不同需要将文库混合??不同的barcode的文库不要混合想,一旦混合,将无法分开


    C ClusterGeneration

    使用cbot仪器 高度自动化,无需人工干预,4小时

    MiSeq、快速HiSeq 2500等少数几种型号的测序仪上也能进行

    步骤:文库模板杂交、桥式PCR、线性化、末端封闭和测序引物杂交

    FC( flow cell)准备就绪后,可以拿到测序仪上真的开始测序了。

    注意:cluster generation最好在测序前先进行。Cluster完成后,越快测序越好。


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