CTCF is required for chromatin loops in the beta ….docVIP

Supplemental Material and Methods 3C Analysis 3C analysis was performed essentially as described (Splinter et al. 2004), using HindIII as the restriction enzyme. Quantitative real-time PCR (Opticon I, MJ Research) was performed with Platinum Taq DNA Polymerase (Invitrogen) and double-dye oligonucleotides (5’FAM + 3’TAMRA) as probes, using the following cycling conditions: 940C for 2 min and 44 cycles of 15 s at 940C and 90 s at 600C. Probe and primer sequences are listed in supplementary Table 2. Chromatin Immunoprecipitation (ChIP) ChIP was performed as described in the Upstate protocol (), except that cells were cross-linked at 2% formaldehyde for 5 minutes at room temperature. Quantitative real-time PCR (Opticon I, MJ Research) was performed using SYBR Green (Sigma) and Platinum Taq DNA Polymerase (Invitrogen), under the following cycling conditions: 940C for 2 min, 44 cycles of 30 s at 940C, 60 s at 550C, 15 s at 720C and 15 s at 750C (during which measurements are taken). Enrichment was calculated relative to Necdin and values were normalized to input measurements. Primer sequences are listed in supplementary Table 3. Antibodies used: Anti-acetyl-Histone H3 (#06-599, Upstate); anti-di-methyl Histone H3 K9/K27 (ab7312, Abcam). Lentivirus production and infection Cre-lentivirus was produced by transient transfection of 293T cells according to standard protocols (Zufferey et al. 1997). 293T cells were transfected with a 3:1:4 mixture of psPAX-2, pMD2G-VSVG (kind gifts of D. Trono, University of Geneva) and a transfer vector construct that is essentially as pRRLsin.sPPT.CMV.GFP.Wpre (Follenzi et al. 2002) but with CMV-Cre instead of CMV-GFP, using poly(ethylenimine) (PEI). After 24 hours medium was refreshed and virus-containing medium was harvested 48 and 72 hours after transfection. After filtration through a 0.45(m cellulose acetate filter, the virus stock was concentrated 1000 times by centrifugation at 19.4K rpm for 2 hours at 100C in a SW28 rotor. Virus stock