4 protocols
Illumina Casava1.7 software used for basecalling. Dirty raw reads were removed from the raw sequence data using a Perl script programmed by BGI Tech. Filter steps were: 1.Remove reads which containing adaptors. 2. Remove reads in which unknown bases are more than 5%. 3. Remove low quality reads (more than half of the bases' qualities are less than 5). 4.Obtain clean reads. Clean reads were mapped to reference genome and genes sequences respectively using SOAP2 (Li, 2009). Mismatches no more than 2 bases were allowed in the alignment. Total base number for each sample should no less than that in the contract. Proportions of clean reads mapped back to genome and genes provide an overall assessment of the sequencing. The calculation of Unigene expression used RPKM method (Reads Per kb per Million reads) (Mortazavi, Williams et al. 2008). Differentially expressed genes were analyzed referring to The significance of digital gene expression profiles (Audic S, Genome Research), and then subjected to GO functional enrichment analysis. Genome_build: RAP-DB Rice Genome Annotation: IRGSP/RAP build 5 Supplementary_files_format_and_content: Tab-delimited .txt files include RPKM values for each sample.
Four-day old Zhongxian 3037 and tog1 seedlings were both divided into two groups and grown in growth chambers (12L:12D cycle at 80% humidity) at 25 ℃ and 30 ℃ respectively for further 18 days.
nucleic acid library construction protocol
Total RNA was extracted using the TRIzol reagent (Invitrogen). Beads with oligo(dT) were used to isolate poly(A) mRNA after total RNA was collected. Fragmentation buffer was added for interrupting mRNA to short fragments. Taking these short fragments as templates, Random hexamer-primer were used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using buffer, dNTPs, RNase H and DNA polymerase I, respectively. Short fragments were purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly(A). After that, the short fragments were connected with sequencing adaptors. And, for amplification with PCR, we selected suitable fragments, as templates, with respect to the result of agarose gel electrophoresis. At last, the library could be sequencing using Illumina HiSeq™ 2000.
For each sample, leaf blades of the fourth leaves were collected, pooled into 100 mg and frozen in liquid nitrogen. Four samples in total were obtained, including Zhongxian 3037 at 25 ℃ and 30 ℃ and tog1 at 25 ℃ and 30 ℃.