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NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat)

NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat)カタログ番号:E7400

カタログ番号

サイズ

濃度

価格

保存温度

E7400S 6 reactions ¥41,500 -20C
E7400L 24 reactions ¥151,200 -20C
E7400X 96 reactions ¥544,400 -20C

製品カテゴリ>グループ

  • NEBNext次世代シーケンサー用ライブラリー調製試薬>前処理試薬

特徴

ヒト/マウス/ラット用 リボソーム RNA 除去キット
バージョンアップにより rRNA 除去効率が向上しました(2020年6月、図 5)
 
特長:
 ・NGS 解析用 ヒト/マウス/ラット RNA サンプル調製の前処理に最適
 ・細胞質 rRNA (5S, 5.8S, 18S, 28S, human ITS, ETS) およびミトコンドリア rRNA (12S, 16S) を除去
 ・10 ng ~ 1 µg の広範囲のトータル RNA 量に対応
 ・FFPE 由来など劣化が進行した RNA サンプルの rRNA も高効率で除去
 ・RNA 精製ビーズ付属キットも別途販売 (#E7405)

 

 図 1:10 ng ~ 1 µg のトータル RNA から rRNA を高効率で除去
 image
Universal human, mouse and rat reference total RNA (1 µg, 100 ng and 10 ng) was depleted of rRNA using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat). RNA-seq libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina followed by paired-end sequencing (2 x 75 bp). 10 Million reads were sampled (seqtk) from each library and reads were identified as ribosomal using mirabait (6 or more shared 25-mers) with rRNA sequences for human baits (5S, 5.8S, 12S, 16S, 18S, 28S, ETS/ITS) or mouse/rat baits (5S, 5.8S, 12S, 16S, 18S, 28S). Levels of rRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. The data represents an average of 3 replicates. The NEBNext rRNA Depletion Kit v2 is efficient at depleting rRNA across species and input amounts.

 

 図 2:非特異的除去なく rRNA だけを除去
 image

Universal human, mouse and rat reference total RNA (1 µg) was depleted of rRNA using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat). RNA-seq libraries were prepared from untreated and depleted RNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina followed by paired-end sequencing (2 x 75bp). 10 million reads were sampled (seqtk) for depleted libraries and 100 million reads for undepleted libraries. GENCODE v27 transcript abundances were estimated using Salmon. Read Counts and R2 values for the linear fit are shown. Transcript expression is consistent after depletion across species.

 

 図 3:インプット量に関わらず、相関した発現レベルを検出

 image
Universal human, mouse and rat reference total RNA (1 µg) was depleted of rRNA using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat). RNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina followed by paired-end sequencing (2 x 75bp). GENCODE v27 transcript abundances were estimated using Salmon. Read Counts and R2 values for the linear fit are shown. Transcript expression correlation is consistent regardless of input amount.

 

 図 4:分解が進んだ FFPE RNA サンプルの rRNA も高効率で除去、発現解析が可能
 image
Human adult normal liver tissue FFPE Total RNA, RIN 2.3 (100 ng and 10 ng) was depleted of rRNA using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (A, B, C) or the TruSeq® Stranded Total RNA Gold kit (A). RNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina® followed by paired-end sequencing (2 x 75 bp). 20 Million reads were sampled (seqtk) from depleted libraries and 200 million reads from undepleted libraries. (A) Reads were identified as ribosomal using mirabait (6 or more shared 25-mers) with rRNA sequences for 5S, 5.8S, 12S, 16S, 18S, 28S and ETS/ITS as baits. Levels of rRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. The data represents an average of 3 replicates and error bars indicate standard error. (B) and (C) GENCODE v27 transcript abundances were estimated using Salmon. Read Counts and R2 values for the linear fit are shown. The NEBNext kit provides superior depletion of rRNA from FFPE (degraded) samples and offers the flexibility of lower total RNA input amounts. Transcript expression correlation is consistent after treatment and regardless of input amount.

 

 図 5:mature rRNA だけではなく pre-rRNA も除去、結果として高効率な rRNA 除去が可能
 

image

Human universal reference total RNA (1 µg) was depleted of rRNA using the original NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E6310) and the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) (NEB #E7400). RNA-seq libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina followed by paired-end sequencing (2 x 75 bp). 10 Million reads were sampled (seqtk) from each library. Reads were identified as ribosomal using mirabait (6 or more shared 25-mers) with rRNA sequences for 5S, 5.8S 12S, 16S, 18S, 28S and ETS/ITS as baits. Levels of rRNA remaining were calculated by dividing matched reads by the total number of reads passing instrument quality filtering. The data represents an average of 3 replicates. NEBNext rRNA Depletion Kit v2 provide superior depletion of rRNA.

 

 図 6:ワークフロー
 

image

 

別途必要な試薬等

【 rRNA 除去に必要な試薬 】

・精製用ビーズ:Agencourt® RNAClean® XP (Beckman Coulter, Inc., Cat. #A63987)の使用を推奨

(またはRNA精製ビーズ付 rRNA 除去キット)

・マグネティックラック

・エタノール

・ライブラリー調製キット など


【 NGS ライブラリー調製に必要なキット 】

・NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (#E7760)

・NEBNext Multiplex Oligos for Illumina (#E7335 など)

キット内容

・NEBNext DNase I

・NEBNext Thermostable RNase H

・NEBNext v2 rRNA Depletion Solution

・RNase H Reaction Buffer

・DNase I Reaction Buffer

・Nuclease-free Water

・NEBNext Probe Hybridization Buffer

 

*保存温度:-20℃

メモ

【2020年12月22日:マニュアルを更新しました】

アダプターライゲーション反応の一部量を修正 (Step 3.10.2、4.10.2、5.10.2、ウェブプロトコール)

関連製品

・NEBNext Poly(A) mRNA Magnetic Isolation Module

・NEBNext Globin & rRNA Depletion Kit (Human/Mouse/Rat)

・NEBNext Globin & rRNA Depletion Kit (Human/Mouse/Rat) with RNA Sample Purification Beads

・NEBNext Ultra II Directional RNA Library Prep Kit for Illumina

・NEBNext Ultra II Directional RNA Library Prep with Sample Purification Beads

・NEBNext rRNA Depletion Kit (Bacteria)