RRBS sequencing of gregarious locust brain sample with short insert library size
Brain tissues of 3-day-old fourth-instar gregarious and solitarious females were dissected and placed in liquid nitrogen. Each sample contained 100 brain tissues. DNA was extracted using the Gentra puregene Kit (Qiagen, USA). Then, 5 μg of genomic DNA was digested with 300 U of the MspI enzyme (New England Biolabs, USA) in 100 μl reactions at 37°C for 16-19 h. After purification, the digested products were subjected to blunt-ending, dA addition and methylated-adapter ligation. To obtain DNA fractions of 40-120 bp and 120-220 bp ranges of MspI-digested products, two ranges of 160-240 bp and 240-340 bp adapter ligated fractions were excised and purified from a 2% agarose gel. Bisulfite conversion was conducted using the ZYMO EZ DNA Methylation-Gold KitTM (ZYMO) following the manufacturer's instructions. The same bisulfite conversion was also conducted for one sample that was not digested by MspI. The final libraries were generated by PCR amplification of 11-13 cycles using JumpStartTM Taq DNA Polymerase (Sigma). After testing using an Agilent 2100 Bioanalyzer (Agilent Technologies) and real time PCR, the libraries were analyzed on HiSeq 2000.