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NEXTflex® Rapid Directional qRNA-Seq™ Kit 链特异性qRNA文库制备试剂盒



NEXTflex® Rapid Directional qRNA-Seq™ Kit能够通过RNA测序,高精度的方向性的分析基因的表达。该试剂盒能够精确的检测转录本的链特异性(>99%),覆盖的均一性、高置信度覆盖率,提高富含CG、低丰度转录本的覆盖率。与Cellular Research 公司联合开发,NEXTflex® Rapid Directional qRNA-Seq Kit生成方向性RNA测序文库与传统的单链RNA测序建库相同,但是引入了特定的分子标签 。


单个RNA的分子标签


NEXTflex® Rapid Directional qRNA-Seq Kit包含一套96种含分子标签的测序接头。每个标签有一个独特的8核苷酸组成。在连接反应中,每个cDNA末端独立、随机的从96种接头中选择和连接一个单个的接头,双末端可以选择的组合则有96×96=9216种 。对于每个克隆测序,双末端reads显示选择的分子标签,连同相邻的cDNA序列。通过这些标签可区分PCR扩增前同一序列的不同的DNA拷贝数,这样即使经过了PCR扩增步骤也能得到较为准确的该序列的起始量。使用分子标签信息的分析为基因表达水平提供一个绝对的、数字化的测量,不需要顾虑大多数RNA测序实验中会出现的扩增偏差。


多重测序


NEXTflex® Rapid Directional qRNA-Seq Kits 包含barcodes,因此不需另外购买barcodes。8次反应的剂盒包括4种barcodes用于多重测序。48 reactions试剂盒有4种不同的版本,每个都包含24种不同的barcodes。多达96种样品可同时进行测序。


1 NEXTflex™ Rapid Directional qRNA-Seq™ Kit.png

没有分子标签的传统RNA-Seq 与带分子标签的NEXTflex® Rapid Directional qRNA-Seq™ 的比较

说明书

产品组分

实验流程

引用文献

相关产品


试剂盒组分

NEXTFLEX® RNA Fragmentation Buffer

NEXTFLEX® First Strand Synthesis Primer

NEXTFLEX® Directional First Strand Synthesis Buffer Mix

NEXTFLEX® Rapid Reverse Transcriptase

NEXTFLEX® Directional Second Strand Synthesis Mix

NEXTFLEX® Adenylation Mix

NEXTFLEX® Ligation Mix

NEXTFLEX® Molecular Index Adapters (1 µM)

NEXTFLEX® Uracil DNA Glycosylase

NEXTFLEX® qRNA-Seq™ Universal Forward Primer (10 µM)

NEXTFLEX® PCR Master Mix

NEXTFLEX® qRNA-Seq™ Barcoded Primers (10 µM)

Nuclease-free Water

Resuspension Buffer


用户自备材料

10 ng – 100 ng mRNA or rRNA-depleted RNA or 10 ng – 10 µg total RNA

100% Ethanol (stored at room temperature)

80% Ethanol (freshly prepared)

2, 10, 20, 200 and 1000 µL pipettes

RNase-free pipette tips

Nuclease-free 1.5 mL microcentrifuge tubes

Thin wall nuclease-free 0.5 mL microcentrifuge tubes

96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) / or / similar

Adhesive PCR Plate Seal (BioRad, Cat # MSB1001)

Agencourt AMPure XP 60 mL (Beckman Coulter Genomics, Cat # A63881)

Magnetic Stand -96 (Ambion, Cat # AM10027) / or / similar for post PCR cleanup

Microcentrifuge

Thermocycler

Heat block

Vortex


2 NEXTflex™ Rapid Directional qRNA-Seq™ Kit.png

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