Tb greentm premix extaqtm 2

Approximately 4 × 10⁶ cells were collected for each ChIP reaction. Cross-linked cells were collected according to the instructions, and ChIP lysate was added for ultrasonic treatment to fragment the chromatin. Then, 50 μL of the solution containing cross-linked chromatin fragments was purified using a ChIP kit (Cell Signaling Technology), and 10 μL of the resulting purified DNA was subjected to 1% agarose gel electrophoresis to observe the size of the DNA fragments. The cell lysates of different treatment groups were combined with YY1 (Proteintech) and IgG (negative control; Abcam) for the ChIP assay, and the bound DNA was purified for quantitative analysis by qPCR. TB Green^TM Premix ExtaQTM II was used to perform qRT–PCR analysis on a LightCycler 480 II instrument (Roche), and the ChIP-enrichment rate was calculated according to the experimental results.

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The first strand of mRNA was reverse-transcribed with 2 μg total RNA using the FastKing gDNA Dispelling RT SuperMix Kit, and real-time quantitative polymerase chain reaction was performed using the Super Real Premix Plus Kit (SYBR Green, Tiangen, Beijing, China). TB Green^TM Premix ExtaQTM II was used for qRT–PCR analysis on a LightCycler 480 II instrument (Roche, Merck). GAPDH was used as an internal reference for lncRNAs and other target genes, and the relative level of gene expression was calculated using the 2^−ΔΔCT method. All primer sequences are listed in Tables S1 and S2.