4 protocols
normalization data transformation protocol
The library products were sequenced using the Illumina HiSeq 2000 Firstly, Low-quality reads that contained more than 30% ‘N’s or over 10% of the sequence with low quality value (quality value <20) per read were omitted from data analysis. Then the sequencing adapters were trimmed off from clean reads. The raw reads were mapped to the reference sequence TAIR9 using SOAPaligner soap2.20(http://soap.genomics.org.cn/index.html) with default parameters. The digested sites were detected from uniquely mapped reads and CNNR sites methylation was determined by a perl script. The regular matching seeking algorithm in perl was used to identify the CNNR sites within the mapped reads, and the cytosines in notarized CNNR sites obtained by MspJI-seq were determined as the methylated cytosines. The WGBS seq data (aerial_tissues_BS_seq_CNNR.gff) generated from GSM399600 Supplementary files (GSM399600_aerial_tissues_BS_seq_alignment_batch-*.gff.gz) was utilized to do comparative study with our MspJI-seq data including sensitivity and specificity analysis. Genome_build: TAIR9 Supplementary_files_format_and_content: ara_rep*_MspJI_site.txt file format: 1.Chromosome; 2.Coordinate for single 'c'; 3.the strand of the single 'c'; 4.tags; 5.consensus sequences type for single 'c'; 6.the real sequence context; 7.three bases sequence context. See 'README.txt' for more information.
sample treatment protocol
arabidopsis leaves were cutted down and cleaned by ddH2O, then they were soaked in ethylalcohol for 1-2 minutes and dried by absorbent paper before extract preparation.
nucleic acid library construction protocol
1.5µg genomic DNA was digested at 37°C for 16h by 12U MspJI enzyme (NEB) in the presence of 0.8µM double-stranded DNA activator (Invitrogen) in a 30µl volume. The digestion system was optimized for the Arabidopsis genome from the original NEB protocol. By running the digested DNA in a 15% native polyacrylamide gel electrophoresis (PAGE), a narrow-band containing all the visible fragments around 28-35bp was excised in reference of 10bp DNA ladder (NEB). DNA was isolated by Crush and Soak Method(Sambrook J: Gel Electrophoresis of DNA and Pulsed-Field Agarose. In Molecular cloning. Volume 2. 3 rd edition. New York: Cold Spring Harbor Laboratory Press; 2001) and purified by ethanol precipitation. Recovered DNA was used to construct sequencing library according to the Illumina Pair-End protocol including procedures of DNA end-repair, ‘A’ base addition, adapters ligation and PCR amplification. Phenol: chloroform extraction and ethanol precipitation were used to purify the products of each process. PCR reaction was fulfilled by JumpStart™ Taq DNA Polymerase (Sigma) for 6 cycles, and its products at length of 148-155bp were recovered from a 2% agarose gel electrophoresis in reference of 50bp DNA ladder (NEB), and purified according to QIAquick gel extraction kit (Qiagen). The obtained Library was analyzed by Bioanalyzer analysis system (Agilent, Santa Clara, USA) before sequencing with Illumina HiSeq2000.
growth protocol
arabidopsis thaliana (ecotype columbia) grow in 28℃ ,2000 lx light for 45 days.