P-GSE42170-1 - P-GSE42170-1
nucleic acid library construction protocol
For mouse TKO ES DNA, cells derived from the Dnmt1-/-, Dnmt3a-/- and Dnmt3b-/- clone #19, gift from M.Okano (Tsumura et al., 2006), were grown on gelatine (no fibroblast feeder cells) in complete ES medium (as in Ficz et al., 2010 ). Before harvesting, the cells were stained by immunofluorescence against 5-methylcytosine and Dnmt1 for potential parental line contaminants (results not shown). Genomic DNA was prepared using the Qiagen DNEasy Blood & Tissue Kit, according to the manufacturer’s instructions. For the TKO ES bisulfite sequencing library, genomic DNA spiked in with a 2kb PCR fragment from M13mp18 (1:10,000 ratio) were fragmented via sonication with a Covaris E220 instrument in a total volume of 70 µl. Methylated adapters (Illumina) were ligated to 250ng of the fragmented DNA with the NEB Next DNA Library Prep Master Mix Set for Illumina, according to the manufacturer‘s instructions, and subsequently BS-converted using Imprint DNA Modification kit (Sigma). The ligated fragments were amplified (15 cycles) using Pfu Turbo Cx Hotstart DNA Polymerase (Agilent) and indexed adapter-specific primers for Illumina – iPCRtagT5 (Quail et al., 2012 ), followed by purification and size-selection using AMPure XP beads (Agencourt). Paired end 100 bp sequencing was performed on an Illumina HiSeq.