14. Analyses by SOLiD (DGE, ChIP-seq, Small RNA-seq)

  Kaori Miyawaki, Chaoyang Cheng, Tomoaki Nishiyama, Tetsuya Kurata




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).



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)