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NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina

NEBNext Single Cell/Low Input RNA Library Prep Kit for Illuminaカタログ番号:E6420

カタログ番号

サイズ

濃度

価格

保存温度

E6420S 24 reactions ¥209,800 -20C
E6420L 96 reactions ¥693,800 -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>Illumina用(DNA, ChIP, mRNA, Small RNA)

特徴

image NEBNext Single Cell/Low Input RNA Library Prep kit
 シングルセル、またはわずか 2 pg RNA よりライブラリー調製

 

特長:    テクニカルノート(英語、PDF)
 ・シングルセル、または微量 RNA (2 pg - 200 ng) からライブラリー調製 
 ・わずか 26 分間のハンズオンタイム、7 時間以内にライブラリー調製
 ・転写量が少ない RNA からも効率的にライブラリー調製
 ・ライブラリーは完全長の mRNA 遺伝子情報を保持
 ・シングルセル(RNA)から cDNA 合成までのモジュールも別途提供 (E6421)

 

 図1:わずか 7 時間でシングルセルからライブラリー調製

image

 

 図2:NEBNext Single Cell/Low Input RNA ライブラリー調製のワークフロー

image

 

 

 図3:シングルセルまたは微量 RNA から高収量のライブラリーを調製

image

  Sequencing libraries were generated from HeLa, Jurkat and M1 single cells or 10 pg of Universal Human Reference (UHR) RNA (Agilent® #740000) with recommended amounts of ERCC RNA Spike-In Mix I (Thermo Fisher Scientific® #4456740). The NEBNext Single Cell/Low Input RNA Library Prep Kit, or the SMART-Seq v4 Ultra® Low Input RNA Kit for Sequencing (Clontech® #634891) plus the Nextera® XT DNA Library Prep Kit (Illumina® #FC-131-1096) were used. Error bars indicate standard deviation for 6-11 replicates. For the NEBNext workflow ~80% of the cDNA was used as input into sequencing library preparation, and libraries were amplified with 8 PCR cycles. For the SMART-Seq®v4/Nextera XT workflow, as recommended, 125 pg of cDNA was used as input in sequencing library preparation and 12 PCR cycles were used for amplification. Error bars indicate standard deviation for 6-11 replicates.  

 

 

図4:転写量が少ない RNA も確実に検出

image

  Sequencing libraries were generated from Jurkat single cells (6 replicates) using the NEBNext Single Cell/Low Input RNA Library Prep Kit, or the SMART-Seq v4 Ultra® Low Input RNA Kit for Sequencing (Clontech® # 634891) plus the Nextera XT DNA Library Prep Kit (Illumina® #FC-131-1096). Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). TPM = Transcripts per Kilobase Million. Each dot represents the number of transcripts identified at the given TPM range, and each box represents the median, first and third quartiles per replicate and method. Salmon 0.6 was used for read mapping and quantification of all GENCODE v25 transcripts. Panels show the number of transcripts detected within the following TPM ranges: 1-5, 5-10, 10-50 and >50 TPM. Increased identification of low abundance transcripts is observed with the NEBNext libraries.  

 

 

図5:転写産物の全領域を均一にカバー

image

  Sequencing libraries were generated from HeLa, Jurkat and M1 single cells, or 10 pg of Universal Human Reference (UHR) RNA (Agilent® #740000) with recommended amounts of ERCC RNA Spike-In Mix I (Thermo Fisher Scientific #4456740). The NEBNext Single Cell/Low Input RNA Library Prep Kit, or the SMART-Seq v4 Ultra® Low Input RNA Kit for Sequencing (Clontech® # 634891) plus the Nextera XT DNA Library Prep Kit (Illumina® #FC-131-1096) were used. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). Gene body coverage shown is an average of four replicates and was calculated using Picard tools. The global view of the 5´ to 3´ coverage of the RefSeq transcripts reveals both consistency across different sample types and uniformity across the transcript length in the NEBNext libraries.  

 

 

図6:シングルセルから信頼性の高いライブラリーを調製

image

  Sequencing libraries were generated from HeLa, Jurkat and M1 single cells, or 10 pg of Universal Human Reference (UHR) RNA (Agilent® #740000) with recommended amounts of ERCC RNA Spike-In Mix I (Thermo Fisher Scientific® #4456740). The NEBNext Single Cell/Low Input RNA Library Prep Kit, or the SMART-Seq v4 Ultra® Low Input RNA Kit for Sequencing (Clontech® # 634891) plus the Nextera XT DNA Library Prep Kit (Illumina #FC-131-1096) were used. Libraries were sequenced on an Illumina NextSeq® 500 using paired-end mode (2x76 bp). Error bars indicate standard deviation for 6-11 replicates. TPM = Transcripts per Kilobase Million. Salmon 0.6 was used for read mapping and quantification of all GENCODE v25 transcripts. A higher number of transcripts were detected in the NEBNext libraries for all sample types.  

 

別途必要な試薬

アダプターオリゴ

・DNA 精製ビーズ [SPRIselect beads または AMPURE XP Beads (Beckman Coulter, Inc.)を推奨]など

キット内容

NEBNext Single Cell RT Primer Mix*

NEBNext Single Cell RT Buffer*

NEBNext Template Switching Oligo*

NEBNext Single Cell RT Enzyme Mix*

NEBNext Single Cell cDNA PCR Master Mix*

NEBNext Single Cell cDNA PCR Primer*

NEBNext Cell Lysis Buffer*

Murine RNase Inhibitor*

NEBNext Bead Reconstitution Buffer*

NEBNext Ultra II FS Enzyme Mix

NEBNext Ultra II FS Reaction Buffer

NEBNext Ultra II Ligation Master Mix

NEBNext Ligation Enhancer

NEBNext Adaptor Dilution Buffer

NEBNext Ultra II Q5 Master Mix

TE Buffer

Nuclease-free Water

*cDNA合成に使用する試薬。別途モジュールとして販売(#E6421)。