GSM3693431: ICC283 Genotype control 2; Cicer arietinum; Bisulfite-Seq
The genomic DNA for respective samples was isolated from frozen leaf tissues using Qiagen DNeasy Minikit (Qiagen). The quality check and quantification of the genomic DNA samples was performed by Nanodrop Spectrophotometer (Thermo Scientific) and Qubit Fluorimeter (Life Technologies). Agarose gel electrophoresis was also conducted to determine the quality of respective genomic DNA samples. The genomic DNA isolated from chickpea leaf tissue at shoot apical meristem stage was further processed for bisulphite sequencing. To perform bisulphite sequencing Ovation® RRBS Methyl-Seq Library System was used for restriction digestion using MspI enzyme according to manusfature's protocol described briefly below. This approach utilizes the methylation insensi¬tive restriction enzyme MspI, which recognizes CCGG. As a result of partial fragmentation during bisulfite conversion, PCR, and efficiency of cluster generation, only a subset of these fragments, typically under 300 bp in length, are sequenced Ovation® RRBS Methyl-Seq Library System requires 100 ng of DNA for RRBS. The respective genomic DNA samples for both genotypes were fragmented via sonication technique to a smaller size base pairs of approximately 100-300 bp, which were further end repaired and in the end TruSeq-methylated adapters were ligated to the DNA fragments. For bisulphite conversion of unmethylated C's EZ DNA Methylation-GoldTM kit (Zymo Research Corporation, CA, USA) used, using approximately 500 ng of adapter-ligated DNA fragments were used according to manufacturer's protocol. The library quality was analysed after desalting, size selection, and PCR amplification was performed. The qualified bisulphite converted libraries were sequenced on the HiSeq 3000 system (Illumina Inc) for 150 cycles in the paired-end mode to achieve more than 30X genome coverage for each sample and processed sequence data (after removal of reads containing adaptor sequences and low-quality reads).  The quality control of the pair end reads was performed by CLC genomics workbench 11.0.1(Qiagen) NGS core tools to remove Illumina adaptors and low quality reads. RRBS Reduced representation bisulphite sequencing was utilized for this study.The qualified bisulphite converted libraries were sequenced on the HiSeq 3000 system (Illumina Inc) for 150 cycles in the paired-end mode to achieve more than 30X genome coverage for each sample
GEO Accession
GSM3693431