ChIP was performed as previously described64 (link). The used antibodies include CTCF (Millipore, cat. no. 07–729), POLR2A (Cell Signaling, cat. no. 14958) and immunoglobulin-G from rabbit serum (Sigma, cat. no. 15006). qPCR was performed using a Power SYBR Green kit (Invitrogen, cat. no. 4368577) with signals detected by a ViiA7 System (Life Technologies). ChIP-seq libraries were prepared using Illumina’s TruSeq ChIP sample preparation kit (Illumina, cat. no. IP-202–1012) according to the manufacturer’s specifications, with the addition of size selection (left side at 0.9×, right side at 0.6×) using SPRIselect beads (Beckman Coulter, cat. no. B23318). Library size was determined (average 351 bp, range 333–372 bp) using the Agilent Bioanalyzer 2100, followed by quantitation using real-time PCR using the KAPA Library Quant Kit for Illumina (KAPA Biosystems, cat. no. KK4835). Libraries were then pooled and sequenced (1 × 75 bp) on the Illumina NextSeq 500 platform according to the manufacturer’s instructions. Bclfastq2 v 2.15.04 (default parameters) was used to convert reads to fastq. Primers used for ChIP-qPCR are listed in Supplementary Table 4 .
Immunoglobulin g from rabbit serum
Manufactured by Merck Group
Immunoglobulin-G from rabbit serum is a purified protein fraction containing polyclonal antibodies of the IgG class, derived from the serum of rabbits. The core function of this product is to provide a source of IgG antibodies for use in various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Truseq chip sample preparation kit, by Illumina (1 mentions)
Viia 7 system, by Thermo Fisher Scientific (1 mentions)
Power sybr green kit, by Thermo Fisher Scientific (1 mentions)
Spriselect beads, by Beckman Coulter (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Ab20920, by Abcam (1 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Tcs sp5 aobs, by Leica (1 mentions)
2 protocols using immunoglobulin g from rabbit serum
1
ChIP-seq Library Preparation and Sequencing Protocol
2
Visualizing PCBUS Morphology and Bacterial Localization
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To examine PCBUS morphology, hematoxylin-eosin (HE) staining was performed as previously described by Feldman and Wolfe (2014) (link). Immunofluorescence staining was used to detect and localize the bacteria in PCBUS, and staining was conducted as previously described for histological slices (de Buhr et al., 2019) (link). Staphylococcus aureus were stained with a rabbit anti-S. aureus antibody (IgG: stock 4 mg/mL, ab20920, 1:100; Abcam). Antibody was diluted in blocking buffer (1% BSA, 5% goat serum, 2% cold water fish gelatin, 0.05% Tween 20, 0.05% Triton X-100 in 1× Tris-buffered saline). Immunoglobulin G from rabbit serum (Sigma I5006, 1.16 mg/mL) was diluted 1:29 as isotype control. As secondary antibody, Alexa Fluor 488 anti-rabbit (1 mg/mL; Thermo Fisher Scientific) was used 1:500 in blocking buffer. Bacteria were visualized using immunofluorescence microscopy and recorded using a Leica TCS SP5 AOBS confocal inverted-base fluorescence microscope with HCX PL APO 40× 0.75 to 1.25 oil immersion objective. The settings were adjusted using isotype control antibodies in separate preparations.
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