title: GSM1632869: iNKT WT BR1 CMS-Seq Input; Mus musculus; OTHER
library_construction_protocol: Vα14i NKT cells were isolated by FACS and DNA was isolated using the PureLink genomic DNA mini kit (Life technologies). DNA was fragmented to an average size of 200 bp using the Adaptive Focused Acoustics Covaris S2 instrument. Library preparation, bisulphite treatment and immunoprecipitation of CMS (5hmC)-enriched DNA were performed as previously described 4,5. Briefly, fully unmethylated l (lambda) DNA (Promega) was spiked into the mouse genomic DNA at a ratio of 1:200. DNA was end-repaired using the End Repair (Illumina Epicentre) and A-tailed (using Klenow fragment, NEB). Ligation of methylated adapters (NeBNext Multiplex Oligos for Illumina, NEB) was performed using Quick Ligase Kit (NEB). DNA was bisulphite-treated using the MethylCode Bisulphite Conversion Kit (Life Technologies). 1% of bisulphite-converted DNA was kept as input and the rest was used for immunoprecipitation with anti-CMS antibody. The anti-CMS recognizes 5hmC that upon bisulphite treatment is converted to cytosine-5-methylenesulfonate (CMS), enabling thus the enrichment of 5hmC containing DNA fragments4. The immunoprecipitated DNA was amplified using Kapa HiFi Uracil+ (Kapa Biosystems).
attributes.tag: GEO Accession
attributes.value: GSM1632869