title: GSM1633774: 5hmC_SC_seq_MEF; Mus musculus; MeDIP-Seq
library_construction_protocol: Sheared gDNA was enriched using Active motif rabbit polyclonal against hydroxymethylation (cat#39769). prior to enrichment through magnetic IgG beads (Dynabeads protein G (Invitrogen #100-03D). Following a series of buffer washes the DNA is then released through proteinase K digestion of the antibody and the DNA is subsequently cleaned up using Qiagen QIAquick PCR purification kits (Qiagen) and eluting in 20ul purified water. Enriched DNA was then subjectedto 10 cycles of whole genome amplification using using an enhanced amplification kit optimised for next generational sequencing (Sigma-Aldrich SeqPlex DNA Amplification Kit). Individual libraries were generated from 100ng each of 5hmc enriched and input DNA for each sample, using the Ion XpressPlus Fragment Library Kit (life Technologies™). The DNA was end repaired, purified and then ligated to Ion-compatible barcoded adapters (Ion Xpress™ Barcode Adapters 1–96: Life Technologies™), followed by nick-repair to complete the linkage between adapters and DNA inserts. The adapter-ligated library was then amplified (10 cycles) and finally size-selected using two rounds of AMPure XP bead capture to size‐select fragments approximately 100–250 bp in length. An equimolar pool of barcoded libraries was prepared at 100pM; each pool contained an hmc-DIP enriched and corresponding input sample. 8pM of the pooled library was added into an emulsion PCR based template reaction; in this reaction the fragments generated during the library prep were attached to Ion sphere particles (ISPs) and clonally amplified. This process was carried out using the Ion One Touch 2 system and the Ion P1 Template OT2 200 Kit.
attributes.tag: GEO Accession
attributes.value: GSM1633774