Sybr greener kit

RNA was isolated with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. For reverse transcription PCR (RT-PCR), 500 ng of total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The cDNA was used for quantitative PCR using SYBR Green ER Kit (Invitrogen) according to the manufacturer’s instructions. Quantitative PCRs were run and the melting curves of the amplified products were used to determine the specificity of the amplification. The threshold cycle number for the genes analyzed was normalized to GAPDH and HPRT. Sequences of the primers used are listed in Supplementary Table 4 and primers for human RANBP6 (PPH13358B), ERBB2 (#PPH00209B-200), and ERBB3 (#PPH00463B-200) are from Qiagen.

For ChIP-qPCR analysis, 1 × 10⁷ ESCs transduced with BirA and linearized Smad7 constructs were used. The immunoprecipitated DNA was isolated using the protocol described for Bio-ChIP and was analyzed by quantitative PCR (qPCR) using the SYBR GreenER kit (Invitrogen, Waltham, MA, USA). For input samples, 250 µL of SDS Elution Buffer was added into 20 µL of the sheared chromatin. The samples were incubated at 65 °C overnight to reverse cross-linking. DNA fragments were purified with the QIAquick PCR Purification Kit and eluted with 40 µL of H₂O. Quantitative PCR was performed with approximately 2% of ChIP sample. The amount of each amplification product was determined relative to a standard curve, and fold enrichment was calculated by comparison of amplified product from the bioChIP sample and ChIP samples from BirA containing ESCs. The amount of immunoprecipitated DNA was calculated relative to input. Primer pairs for quantitative ChIP-PCR were designed using ±150 bp genomic sequence information specific to the predicted target loci to generate 100 bp to 200 bp amplified products. Primers targeting mouse promoter sequences were used for qPCR analysis. Primer sequences are listed in Table S2.

Each ChIP experiment was performed in at least three independent biological samples and performed as previously described [24 (link)]. Briefly, 1 × 10⁶ GBM CSCs, over-expressing either ASCL1 or GFP as control, were cross-linked with 1% formaldehyde for 10 min at r/t and the reaction was quenched by glycine at a final concentration of 0.125 M. Cells were lysed in lysis buffer and chromatin was sonicated. Fifty μg of each sonicated chromatin sample were incubated o/n at 4 °C with the following antibodies: anti-IgG (Santa Cruz) and anti-MASH1 (Ascl1; BD Pharmingen). Immunoprecipitated DNA was analyzed by qPCR by using SYBR GreenER kit (Invitrogen). Values were normalized to those obtained with a non-immune serum and divided by input. The data shown represent triplicate qPCR measurements of the immunoprecipitated DNA. The data are expressed as (‰) express 1/1000 of the DNA inputs. All ChIP experimental values were normalized to those obtained with the relative input sample.

3C-qPCR assays were performed as previously described^{69 (link)} with slight modifications. Briefly, the assays were performed on PC3 cells overexpressed with HOXC5 or control GFP. 1 × 10⁷ nuclei were cross-linked with 1% formaldehyde, digested using DpnII and ligated as described^{69 (link)}. DNA was then reverse cross-linked and purified by extraction with phenol/chloroform, followed by precipitation with ethanol. The DNA concentration of the 3 C libraries was determined using Picogreen fluorescence assay (Invitrogen). 3C-qPCR reactions were performed by the Sybr-Greener Kit (Invitrogen), and the chromatin interactions were normalized by loading control. On the basis of the 4C-seq data, we selected a region in the hTERT coding region with background levels of interaction, with a similar genomic distance from the hTERT bait region as the experimental interaction, to serve as a control interaction. The primers used for 3c-qPCR are listed in Supplementary Table 2.

Two micrograms of sonicated DNA was denatured at 95 °C for 10 min, immediately cooled on ice for 10 min, and diluted in 500 μl of IP buffer (10 mM Na-Phosphate pH 7.0 140 mM NaCl 0.05% Triton X-100), 50 μl as input. Then, add 10 μl of anti-5mC (#28692; Cell Signaling Technology) or anti-5hmC (#39769; active motif; Cell Signaling Technology), IgG (#2729, Cell Signaling Technology) as control. After 2 h incubation at 4 °C with overhead shaking, 30 μl of 50% protein A/G-Sepharose magnetic beads were added and incubated for another 2 h. Put the tube on the magnetic beam for 5 min and discard supernatant. After five washes with IP buffer, DNA was eluted with Proteinase K for 2 h, then purified using the QIAQuick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions. DNA was analyzed by quantitative real-time PCR by using a SYBR GreenER kit (Invitrogen). Primers sequences are provided in Table 1. Fold enrichment was calculated as follows: %Input = 10% × 2^(CT^input−CT^sample).