2012年9月26日水曜日

LIMprep: a highly-sensitive multiplex library preparation method for low amount DNA (English version)

I established LIMprep (Ligation-based Illumina Multiplex library PREParation): a highly-sensitive multiplex library preparation method for low amount DNA. LIMprep requires 4-6 cycles of PCR enrichment for 10 ng DNA, although previous methods require 10-15 cycles of PCR enrichment.  For 100 pg DNA, LIMprep require 10 cycles of PCR enrichment. LIMprep is advantage in quantitative performance. Please see the spec of LIMprep. The protocol of LIMprep is available via NipponGenetics. If you interest, please contact them at first.


*Relation to Single-cell RNA-seq*
We established novel whole-transcript amplification method. After amplification, we got ng level of cDNA. Amplified cDNA was prepared to library DNA with the  LIMprep method. 

*Confirmation by other group*
Sequence service unit already use LIMprep for ChIP-seq. Their sequence was pretty good. 

2012年9月13日木曜日

Memo: Structure of amplified cDNA of Smart-Seq


5’ AAGCAGTGGTATCAACGCAGAGTACATGGGNNNNNNNNNNBAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGTACTCTGCGTTGATACCACTGCTT 3’
3’ TTCGTCACCATAGTTGCGTCTCATGTACCCNNNNNNNNNNVTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCATGAGACGCAACTATGGTGACGAA 5'


デフォルトSmart-Seqの全シーケンスリード数の(完全マッチで)20-25%がWTA adaptor sequence 由来でした。