E-MTAB-7307:treatment_2_p
NextSeq 500 paired end sequencing; DNA-seq of 5fC enriched fragments based
DNA-seq of 5fC enriched fragments based
treatment_2_p
mESCs (E14 p4, ~4 x 10^6) were collected by centrifugation of cell pellets and stored at -80 °C for up to 2 months. DNA was isolated according to the manufacturer's instructions (Qiagen DNeasy blood and tissue kit) DNA was sonicated using the covaris M220 ( Peak power = 50, Cycles/Burst = 200, Duty Factor = 20, Duration = 175 seconds, Temperature range = 18–25 °C ) - DNA was biotinylated using the synthesised c-linker which covalently binds any 5fC bases (McInroy, G. R., Raiber, E. A. & Balasubramanian, S. Chemical biology of genomic DNA: minimizing PCR bias. Chemical communications (Cambridge, England) 50, 12047-12049, doi:10.1039/c4cc05107f (2014).) Sonicated DNA ~500 ng (4 mL NH4OAc pH 5, 20 mL 0.5 M p-Anisidine, 4 mL 10 mM c-linker, NFW up to 100 mL). Overnight at 37 - The library underwent End repair, dA tailing and adapter ligation according to the manufacturer's instructions (illumina truseq LT) - Streptavadin bead pulldown was used to enrich for 5fC containing fragments -The DNA libraries were amplified using the NEBNext Ultra II Q5 Master mix and Illumina primers. 30 seconds at 98 °C. 12 cycles of: 10 seconds at 98 °C, 75 seconds at 65 °C, 5 minutes at 65 °C,hold at 4 °C -The DNA was purified using AMpure XP beads .  In parallel each sample was prepared in absence of biotinylation and streptavadin pulldown as a control
UNIVERSITY OF CAMBRIDGE
E-MTAB-7307:treatment_2_p
Experimental Factor: fraction
DNA bound to biotin/steptavidin (containing 5fC bases)