Itaq universal sybr green supermix form

Cells at 80% confluency were treated with TNF-α or PBS (vehicle). ChIP analysis was performed using the Millipore Magna ChIP assay kit protocol (cat# 17-610). For the immunoprecipitation, 2 μg of appropriate antibody was used for each condition and incubated overnight at 4ºC, with lysates from 10⁶ cells. Quantitative real-time PCR (RT-PCR) was performed with the Applied Biosystems 7900HT RT-PCR instrument using the iTaq Universal SYBR Green Supermix form Bio-Rad (cat. # 172-5121).
The primer sequences were as follows:
Intergenic region 8000 upstream IL-6 promoter:
For. 5′- GCTCCTCCATCTGGTGTCAT-3′, Rev.5′-AAATTGGGGGTAGGGTTGTC-3′
IL-6 TSS:
For. 5′-AATGTGGGATTTTCCCATGA-3′, Rev. 5′-AGTTCATAGCTGGGCTCCTG-3′
TNF-α TSS:
For. 5′-ATCGGAGCAGGGAGGATG-3′, Rev. 5′-CCAGCGGAAAACTTCCTT-3′
IκB-α TSS:
For. 5′-GGAAGGACTTTCCAGCCACT-3′, Rev. 5′-GGAATTTCCAAGCCAGTCAG-3′
For the ChIP/re-ChIP experiment, the immunoprecipitated DNA–protein complex was eluted from the beads by incubating with 50 µl of 10-mM 10mM Dithiothreitol (DTT) diluted in Tris-EDTA (TE) buffer at 37ºC for 30 min. The eluted supernatant was diluted 20 times in ChIP dilution buffer from the Millipore Magna ChIP assay kit, and second round of ChIP was performed using the αPRMT6 antibody.

RNA was isolated from 80% confluent cell plates using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). cDNA was prepared from total RNA (1 μg) using the Superscript III First Strand Synthesis System for RT-PCR from Invitrogen (cat. # 18080-051) following the manufacturer's protocol. Quantitative RT-PCR was performed with the Applied Biosystems 7900HT RT-PCR instrument using the iTaq Universal SYBR Green Supermix form Bio-Rad (cat. # 172-5121) with primers for the indicated genes. Primers were designed across exons.
The primer sequences were as follows:
IL-6:
For. 5′-ACTCACCTCTTCAGAACGAATTG-3′, Rev.5′-CCATCTTTGGAAGGTTCAGGTTG-3′
TNF-α:
For. 5′-CCCCAGGGACCTCTCTCTAA-3′, Rev. 5′-TGAGGTACAGGCCCTCTGAT-3′
MCP1/CCL2:
For. 5′-TCTGTGCCTGCTGCTCATAG-3′, Rev. 5′-GCTTCTTTGGGACACTTGCT-3′
COX-2:
For. 5′- TGAAACCCACTCCAAACACA-3′, Rev. 5′-GAGAAGGCTTCCCAGCTTTT-3′
IKB-α (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha):
For. 5′-AGACCTGGCCTTCCTCAACT-3′, Rev. 5′-TGCTCACAGGCAAGGTGTAG-3′
GAPDH (Glyceraldehyde 3-phosphate dehydrogenase):
For. 5′-AGCCACATCGCTCAGACAC-3′, Rev. 5′-GCCCAATACGACCAAATCC-3′