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Home > ÀüÁ¦Ç°º¸±â > NGS °ü·Ã > mRNA-Seq (Single cell) > Ultra low input 3¡¯end RNA-Seq Kit
3¡¯ Differential Expression (DE) ºÐ¼®À» High-throughputÀ¸·Î

Ultra low input 3¡¯end RNA-Seq Kit

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Clontech
635040
SMART-Seq¢ç v4 3' DE Kit
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Clontech
635041
SMART-Seq¢ç v4 3' DE Kit
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  • SMART-Seq¢ç v4 ±â¹ÝÀ¸·Î single-cell ¶Ç´Â 10pgÀÇ ±Ø¼Ò·® total RNA Àû¿ë
  • Àü»çüÀÇ 3¡¯ ¸»´Ü¸¸ ƯÀÌÀûÀ¸·Î ºÐ¼®ÇÏ¿© ÇÕ¸®ÀûÀÎ °¡°ÝÀ¸·Î High-throughput Differential Expression (DE) ºÐ¼® °¡´É
  • ÃÖ´ë 12 Multiplexing Illumina¢ç Library Á¦ÀÛ °¡´É
ÃÖ»óÀÇ °á°ú¸¦ º¸ÀåÇÏ´Â SMART ±â¼ú
´ÜÀϼ¼Æ÷ (Single cell) Àü»çü ºÐ¼®Àº ¡®¼¼Æ÷Áý´Ü (cell population) ³»¿¡¼­ °¢°¢ÀÇ ¼¼Æ÷ÀÇ ¾î¶² À¯ÀüÀÚ ¹ßÇöÀ²À» º¸ÀÌ´ÂÁö¡¯ ¸¦ ÀÌÇØÇÏ´Â µ¥ Áß¿äÇÏ´Ù. SMART-Seq¢ç v4 3¡¯ DE Kit´Â ´ÜÀϼ¼Æ÷ À¯·¡ÀÇ mRNAÀÇ 3¡¯ ¸»´ÜÀÇ ¼­¿­ Á¤º¸¸¦ ¾òÀ» ¼ö ÀÖ¾î À¯ÀüÀÚ ¹ßÇöÂ÷ÀÌ ºÐ¼® (Differential gene Expression (DE) analysis)¿¡ ÃÖÀûÀÌ´Ù. º» Á¦Ç°Àº SMART¢ç¹ý°ú Locked Nucleic Acid (LNA) ±â¼úÀ» ÀÀ¿ëÇÑ SMART-Seq¢ç v4 Å°Æ®ÀÇ Æ¯Â¡À» µµÀÔÇÏ¿´´Ù.
mRNAÀÇ 3¡¯ ¸»´Ü¸¸ ÁýÁßÀûÀ¸·Î ¼­¿­À» ºÐ¼® °¡´ÉÇϸç, º» Á¦Ç° ³» Read2ÀÇ ¹ÙÄÚµå·Î ÀνÄ, »ç¿ëÇÒ ¼ö ÀÖ´Â In-line À妽º°¡ Æ÷ÇԵDZ⠶§¹®¿¡ »ùÇñîÁö Multiplexing NGS Library¸¦ Á¦ÀÛ ¹× ºÐ¼®ÀÌ °¡´ÉÇÏ´Ù. 1 ~ 10.5ulÀÇ RNA volumeÀ» Àû¿ë °¡´ÉÇϸç Á¦ÀÛµÈ cDNA´Â Illumina Nextera¢ç XT DNA Library Preparation Kit*1¸¦ ÀÌ¿ëÇÏ¿© NGS Library Á¦ÀÛ*2ÀÌ °¡´ÉÇÏ´Ù. º» Á¦Ç°À» »ç¿ëÇÏ¿© Á¦ÀÛµÈ NGS Library´Â Illumina¢ç HiSeq¢ç, NextSeq¢ç*3, MiSeq¢ç, and MiniSeq¢â*3¿¡ Àû¿ëÇÒ ¼ö ÀÖ´Ù.

*1: Fragmentation°ú Tagging °úÁ¤À» À§ÇØ Nextera¢ç XT DNA Library Preparation Kit°¡ º°µµ ÇÊ¿ä
*2: Library index´Â ÀÌ·ÐÀûÀ¸·Î ÃÖ´ë 1,152 (=12 x 96)°³±îÁö °¡´É
*3: NextSeq¢ç, MiniSeq¢â ½ÃÄö¼­·Î ½ÃÄö½ÌºÐ¼®À» Àû¿ëÇÒ °æ¿ì, »ùÇÿ¡ Control DNA (PhiX) Ãß°¡ ÇÊ¿ä


±×¸² 1. SMART-Seq¢ç v4 3' DE Kit¸¦ ÀÌ¿ëÇÑ cDNA ÇÕ¼º°ú Library Á¦ÀÛ
cDNA (black) is synthesized with a blocked (black star) and modified oligo(dT) primer that adds sequences for subsequent amplification and analysis-an in-line index (magenta), part of the Illumina read primer 2 sequence (RP2, yellow), and the SMART IIA sequence (green). The SMART IIA sequence is used as a priming site during cDNA amplification, the Illumina RP2 sequence is used as a priming site during library amplification, and the in-line index is used for demultiplexing pooled samples during analysis. The process works as follows: first, the template for SMARTScribe reverse transcriptase switches from the mRNA (blue wavy line) to the SMART-Seq v4 Oligonucleotide (green). After reverse transcription, the full-length cDNA is amplified by PCR with blocked Primer IIA oligonucleotides. After cDNA amplification, the presence of the in-line index (magenta) allows for pooling of up to 12 samples. The pooled samples are tagmented and Illumina Nextera read primer 1 and 2 sequences are added by the Nextera Tn5 transposon (TnRP1 and TnRP2, orange and purple respectively). The 3' ends of the original mRNA are captured by selective PCR with primers for the TnRP1 and RP2 sequences. Other products of the transposon-based reaction are not amplified, either because there are no primer sites for amplification or because of suppression PCR. Cluster generation (pink and dark purple) and indexing sequences (light blue and dark blue) are added during this PCR stage to generate a library ready for sequencing on an Illumina platform.

Sample ID

Pool

In-line Indexes

i1

i2

i3

i4

i5

i6

i7

i8

i9

i10

i11

i12

mtRNA(%)

3.3

3.1

3.6

1.7

3.9

4.1

1.8

2.5

2.8

5.5

4.1

2.7

4.8

rRNA(%)

0.6

1.2

0.7

1.1

0.4

0.4

0.3

0.3

0.7

1.2

0.5

0.1

0.6

Uniquely mapped reads(%)

69

68

68

70

68

71

71

72

68

69

69

74

69

Total mapped reads(%)

97

96

98

97

97

98

98

98

96

97

96

98

98

Number of reads(M)

21.6

2.3

5

1.3

2.2

2

1.9

0.8

1.3

0.6

0.2

1.5

1.6


Ç¥ 1. K562 single cell·ÎºÎÅÍ Á¦ÀÛµÈ pooled libraryÀÇ mapping read (%)
K562 cells were diluted to one cell/¥ìl in PBS buffer and twelve single cells were isolated, checked via optical microscopy, lysed, and subjected to cDNA synthesis. The pooled libraries were sequenced on an Illumina MiSeq instrument with 47 bp for read 1 and 26 bp for read 2. The pooled libraries were demultiplexed based on the in-line barcode sequence from read 2. All libraries were mapped with STAR v.2.3.0.1 (Dobin et al. 2013) against the human genome (hg19). The reads map to the genome at a high rate (>96%) with a small proportion mapping to rRNA or mitochondrial (mt) regions.
Applications
cDNA synthesis for end-capture mRNA-seq for differential expression analysis of single cells.
±¸¼ºÇ° (ÀÚ¼¼ÇÑ ³»¿ëÀº CoA¸¦ ÂüÁ¶Çϼ¼¿ä)
SMART-Seq v4 3' DE Kit Components
SeqAmp¢â DNA Polymerase

Keyword : Single cell,´ÜÀϼ¼Æ÷,½Ì±Û¼¿,single cell analysis,single cell NGS,targeted sequencing,3'DE,3' DE,DE,differential expression,mRNA,mRNA-seq,cDNA,cDNA-seq,NGS,Next Generation Sequencing,SMART,,smart-seq,Rubicon,·çºñÄÜ

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