4 protocols
nucleic acid sequencing protocol
Sequencing was carried out on an Illumina Genome Analyzer GAIIX using Cluster Generation v2 and 4 chemistries as well as Sequencing by Synthesis Kits v3 and v4. Data collection was performed using Sequencing Control Software v2.5 and 2.6. Real-time Analysis 1.5-1.8 was used for base calling.
nucleic acid extraction protocol
DNA was extracted using a standard phenol choroform procedure.
nucleic acid library construction protocol
MeDIP-Seq was carried out as described previously [21]. Briefly, purified genomic DNA was sonicated to yield 150–600 bp fragments, and adaptors for paired-end sequencing (Illumina, San Diego, CA, USA, http://www.illumina.com) were ligated using NEB Next DNA Sample Prep Reagent Set 1 (New England Biolabs Hitchin, UK, http://www.neb.com). Immunoprecipitations (IPs) were carried out in triplicate using 500 ng DNA per sample, 1.25ug anti-5-methylcytosine (anti-5mC) antibody (Eurogentec, Southhampton, UK, http://www.eurogentec.com), and 10 ul Dynabeads coupled with M-280 sheep anti-mouse IgG (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). The three IPs were pooled and amplified for 12 cycles with adapter specific primers, run on a 1% agarose gel, and fragments ranging between 300 and 500 bp in size were cut out and purified using the Gel Elution Kit (Qiagen, Crawley, UK, http://www.qiagen.com) before cluster generation and sequencing.
growth protocol
ES, TS, XEN, and EpiSC stem cell lines were cultured under standard conditions (see Supporting Information in the publication). Cell lines used were: ES, lines J1, E14, KH2, and PGK12.1 (a kind gift of Prof. N. Brockdorff, University of Oxford, U.K.); EpiSC, lines #5 and #9 (both a kind gift of Dr. P. Tesar, Case Western Reserve University, OH) and line PRG (a kind gift of Dr. P. Rugg-Gunn, The Babraham Institute, Cambridge, U.K.); TS, lines Rs26, GFP, and B6 (kind gifts of Dr. J. Rossant, Hospital for Sick Children, Toronto, Canada and Dr. A. Erlebacher, NY University School of Medicine, New York, USA); and XEN, lines IM8A1 (Dr. J. Rossant), #16, and E4 (Dr. P. Rugg-Gunn).