N lauroylsarcosine

Bacteria cells were grown in LB broth overnight, transferred to fresh LB medium at 1%, and incubated until they reached the mid-log phase of growth. These cells were collected by centrifugation at 10,000 × g for 5 min and resuspended in 10 mL of HEPES buffer (10 mM, pH 7.4). The resuspension was ultra-sonicated, centrifuged, and filtered with a 0.22-μm filter membrane. The membrane fraction was collected by ultra-centrifugation at 100,000 × g for 1 h at 4 °C. The pellet was washed with 10 mL of 10 mM HEPES (pH 7.4) and ultra-centrifuged again as described above. The pellet was resuspended in 10 mL of 10 mM HEPES (pH 7.4) containing 2% N-lauroylsarcosine (wt/vol) (Sarkosyl) (Sigma, USA) and incubated at 37 °C for 30 min with shaking. Sarkosyl-treated membranes were centrifuged at 100,000 × g for 1 h at 4 °C and the pellet was resuspended in 50 mM Tris-Cl (pH 8.0) containing 1% (wt/vol) Zwittergent 3–14 (Calbiochem, USA) and 10 mM EDTA.

The chemical reagents used in the study were as follows: deoxyadenosine 5′-triphosphate, [α-³³P] (NEG312H; NEN Research Products, PerkinElmer, Waltham, MS, USA); CaCl₂, NaCl, KCl (Merck, Whitehouse Station, NJ, USA); aminoguanidine hydrochloride, Denhardt's solution, dithiothreitol, N-lauroylsarcosine, polyethylene glycol 300, β-mercaptoethanol, phenylmethanesulfonyl fluoride, S-adenosyl-methionine (Sigma, St Louis, MO, USA); MgCl₂ and (Riedel-deHaёn, Seelze, Germany); formamide (Amresco, Solon, OH, USA); RNA (Roche, Basel, Switzerland); and dextran sulphate (Amersham, Amersham, UK).

The ChIP assay was performed by first crosslinking ~ 10 million cells with 1% formaldehyde at room temperature for 10 min, washing the cells twice with 1 × PBS, and then followed by lysing the cells with 1 mL of Lysis Buffer A (50 mM HEPES, 140 nM NaCl, 1 mM EDTA pH 8.0, 10% Glycerol (Sigma G5150), 0.5% NP-40 (Igepal CA-630, Sigma I3021), 0.25% Triton X-100), centrifuging the cells at 1,350 × g for 5 min at 4 °C, and re-suspending the pellet with 1 mL of Lysis Buffer B (10 mM Tris-HCl pH 8.0, 200 nM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA), centrifuging the cells at 1350 × g for 5 min at 4 °C, and a final resuspension of the pellet with 300 µL of Lysis Buffer C (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA, 0.1% Sodium deoxycholate (Sigma D6750), and 0.5% N-lauroylsarcosine (Sigma L5777)^{62 (link)}. All the lysis buffers included the cOmplete, Mini Protease Inhibitor Cocktail (Sigma 11836153001). The chromatin was sheared by using a Covaris S220 instrument. The pull-down was performed using 10 µg of CTCF antibody (1:1000 dilution, Millipore Sigma 07–729). The reads were aligned to the mm9 human genome using the Bowtie2 tool^{59 (link)}. The ChIP-seq data was analyzed using the HOMER suite^{63 (link)}. The motif orientations were determined by the FIMO software^{64 (link)}, using the CTCF motif position weight matrix (MA0139.1) from the JASPAR database^{65 (link)}.

HeLa cells were split in 24 well plates at a concentration of 75 000 cells/well and transfected 12 hours later with 10 nM dsiRNA combined with lipofectamine 2000 (Invitrogen) as recommended by the supplier. dsiRNA were transfected again 24 and 48 h after the first transfection. The culture was maintained for a total of 4 days after the first transfection. The culture medium was removed and the cell monolayer was washed carefully with 500 µl PBS. Cells were stained for 30 min at RT by the addition of 200 µl of a solution containing 5 mg/ml of methylene blue in 50% ethanol. The plate was carefully and extensively washed with water until no blue stain remained in the water. The plate was air dried completely. 500 µl of a PBS solution containing 10 mg/ml N-lauroyl sarcosine (Sigma L-5125) was added to each well. Lysis was performed for 1 h at RT. 100 µl of each lysate was used to measure absorbance at 595 nm (A515 nm = control), corresponding to methylene blue incorporation and cell content.

Metagenomic DNA was obtained from cecal content of fasted SPF mice after mechanical lysis followed by purification according to a published protocol [60 (link)] modified as follows: cecal content in 600 μl stool DNA stabilizer (Stratec Biomedical AG) was transferred into a 2-ml screw-cap tube containing 500 mg zirconia/silica beads (0.1 mm; BioSpec Products), 250 μl 4 M Guanidinethiocyanate (Sigma-Aldrich, Germany), and 500 μl 5% N-lauroylsarcosine (Sigma-Aldrich, Germany). Samples were mixed and incubated for 60 min at 70 °C with constant shaking, and bacterial cells were disrupted by mechanical lysis using a FastPrep®-24 (three times, 40 s, 6.5 m/sec) (MP Biomedicals) fitted with a cooling adaptor. After addition of 15 mg polyvinylpolypyrrolidone (PVPP, Sigma-Aldrich, Germany), the suspension was vortexed and centrifuged (3 min, 15,000×g, 4 °C). The supernatant (500 μl) was transferred into a new Eppendorf tube, mixed with 5 μl RNase (VWR International, stock concentration 10 mg/ml) and incubated for 20 min at 37 °C with constant shaking. Genomic DNA was purified using NucleoSpin® gDNA columns (Macherey Nagel GmbH & Co. KG, Germany) following the manufacturer’s instructions. DNA quantity and quality were measured with a NanoDrop® instrument (Thermo Fisher Scientific Inc., Germany).