GSM3884760: ChIP-PR - Progesterone – siCEBPa; Homo sapiens; ChIP-Seq
ChIP protocol: Chromatin Immunoprecipitation (ChIP) in cultured cells: ChIP assays were performed as described (Strutt and Paro, 1999) using anti-PR (Santa Cruz, H190); anti-C/EBPα (Cell Signaling, 8178S); anti-BRG1 [EPNCIR111A] (Abcam, ab110641), anti-LSD1 (Abcam, ab17721); anti-Topo2α (Santa Cruz, sc-3659), anti-P300 (Santa Cruz, sc-584x and sc-585x), anti SRC3 (Santa Cruz, sc-9119x). Quantification of chromatin immunoprecipitation was performed by real time PCR using Roche Lightcycler (Roche). The fold enrichment of target sequence in the immunoprecipitated (IP) compared to input (Ref) fractions was calculated using the comparative Ct (the number of cycles required to reach a threshold concentration) method with the equation 2Ct(IP)-Ct(Ref). Each of these values were corrected by the human β-globin gene and referred as relative abundance over time zero. Primers sequences are available on request. RNA extraction(RNA-seq): RNA extraction and RT-PCR  Total RNA was prepared and cDNA generated as previously described (Vicent et al., 2006). Quantification of LUC and GAPDH gene products was performed by real time PCR. Each value calculated using the standard curve method was corrected by the human GAPDH and expressed as relative RNA abundance over time zero. Primer sequences for DUSP1, C/EBPα, PGR are available on request. RNA-seq libraries were constructed following the Illumina Truseq Stranded mRNA Sample Prep Kit (RS-122-2101). ChIP-seq libraries were constructed following the NEBNext DNA samples prep kit (Ref.: NEB # E6000S) and PE adaptors. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols. RNA-seq and ChIP-seq. We use the NEBNext Ultra DNA Library Prep Kit for Illumina kit (Ref.:#7370), with amplification by real-timePCR (light cycler) to determine the number of cycles to amplify the library