Iq5 quantitative pcr instrument

The total RNA extracted from thymus organs of both 2-W and 40-W groups was used for fluorescent quantitative PCR analysis (qRT-PCR). The cDNA was synthesized using the PrimeScript RT reagent Kit (TaKaRa). The qRT-PCR was performed with SYBR® Premix Ex Taq TM II (TaKaRa) following the protocol provided by manufacturer on an IQ5 quantitative PCR instrument (Bio-Rad Laboratories, Hercules, CA), and programmed as follows: 95°C for 5 min; 40 cycles of 95°C for 10 s, 60°C for 30 s, 72°C for 30 s; and 72°C for 5 min. The 20 μL RT-qPCR mixture included: 10 μL of SYBR Premix Ex Taq II (2 ×), 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 1 μL of template DNA, and 7 μL of ultrapure water. The reference gene, β-actin was used as an internal expression control. All samples were run in triplicate, and 2^−ΔΔCt method was used to calculate the transcriptional level of the selected DEGs. The primer sequences (Table S3) for the 8 DEGs (TLR1, TLR4, TLR5, CD40, AP-1, IL8, TACI, and PIGR) were designed using online NCBI/Primer-BLAST software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/).

Limb buds from E 11.0 embryos were dissected, and the mesenchymal cells were transfected with the previously described LncRNA-HIT siRNAs. 24 H after transfection, qChIP assays were performed as described [106 –108 (link)], using an anti-acetyl histone H3 (lys27) antibody (Cat. 07–360, Millipore, Billerica, MA) or mouse IgG (Diagenode: C15400001) as a negative control. DNA sequences specific to H3K27ac peaks proximal to LncRNA-HIT -associated peaks were identified using an overlay of the E 10.5 limb bud H3K27ac ChIP-seq dataset [63 (link)] and the UCSC Genome Browser. Primers were selected to amplify the DNA regions containing the H3K27ac peaks using Primer3 (simgene.com) (S4 Table). ChIP-enriched H3K27ac fragments were quantified by qPCR using an IQ5 quantitative PCR instrument (BioRad, Hercules, CA) as described [107 (link)–108 (link)].