14. Analyses by SOLiD (DGE, ChIP-seq, Small RNA-seq)
Kaori Miyawaki, Chaoyang
Cheng, Tomoaki Nishiyama, Tetsuya Kurata
Introduction
The SOLiD system is a one of highly accurate,
massively parallel next-generation sequencer (Holt and Jones, 2008). By using SOLiD system, several applications
including genome resequencing, digital gene expression (DGE), small
RNA-sequencing, whole transcriptome analysis, Chromatin
immunoprecipitation-sequencing (ChIP-seq), and genome methylation analysis. Now in version 3, we can get 100-600 million
short reads (25-50 nt) which constitute total 2.5-30 Gbase sequence (http://www3.appliedbiosystems.com/AB_Home/index.htm)
with greater than 99.94% basecalling accuracy.
Materials
and Methods
For DGE, 5-10 mg
total RNA is required. Total RNA is
routinely extracted with RNeasy Plant Mini Kit (QIAGEN). Quality should be checked by Bio Analyzer
(Agillent). Before preparation of DGE
library, polyA+ fraction was enriched by FastTrack MAG micro mRNA Isolation Kit
(Invitrogen). If you want to know the
procedure for DGE library, please contact T. Kurata (tekurata@nibb.ac.jp) or M. Hasebe
(mhasebe@nibb.ac.jp).
For small RNA-seq,
500 ng total RNA including small RNA fraction. We used miRNeasy Mini Kit with QIAshredder (QIAGEN). Quality should be checked by Bio Analyzer
(Agillent). Small RNA-seq library was
prepared by according with kit manual (SOLiD Small RNA Expression Kit;
AB/Ambion).
For ChIP-seq, the short DNA fragments (100-400 ng)
after ChIP (see chapter 11.8) was used for fragment library construction. ChIP-seq libraries were prepared by by
according with SOLiD fragment library protocol (AB).
“It is important to precisely quantify library
concentration by QPCR at adaptor region
(Meyer et al., 2008)”.
Following emPCR (emulsion PCR), beads enrichment,
3’end modification, beads deposition, sequence run have been done by according
with SOLiD manual (http://www3.appliedbiosystems.com/AB_Home/index.htm). After base colling, mapping to reference
moss genome (480 Mb-nuclear+mitochondria+chloroplast) was conducted by using
Corona_lite mapping tool (AB) in HP xw8600/CT Workstation (2CPU, 64GB;
Hewlett-Packard Development Company).
References
Holt and Jones,
The new paradigm of flow cell sequencing.
Genome Res. 18, 839-846 (2008)
Meyer et al.,
From micrograms to picograms: quantitative PCR reduces the
material demands of high-throughput sequencing.
NAR 36, e5 (2008)