Ultrapure rna kit

The LMH cells (70–80% confluence) were stimulated with FST (10⁻⁴), EPS (0.1 mg/mL), and L-Tyr (0.25 mmol/L), respectively. Total RNA was extracted using the Ultrapure RNA Kit (CW0581M, CoWin Biosciences, Beijing, China) and the reverse transcription reaction was performed using PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) following the manufacturer's protocol. After mixing the extracted RNA with ChamQ Universal SYBR qPCR Master Mix (Q711-02/03, Vazyme Biotech Co., Ltd., Nanjing, China) and specific primers, the qRT-PCR analyses were performed on a CFX96TM (Bio-Rad, Richmond, CA, USA). Primers used for the qRT-PCR analysis (Han et al., 2015 (link)) are shown in Supplementary Table 4.

Total cellular RNA was extracted with an Ultrapure RNA kit (CW0581M; CoWin Biosciences, China) according to the manufacturer’s instructions. Total RNA (500 ng) was reverse transcribed into cDNA (Toyobo) and then analyzed by real-time PCR with FastStart Essential DNA Green Master (Roche). The following primers were used: for human IFN-β, the sense primer was 5′-AGGACAGGATGAACTTTGAC-3′ and the antisense primer was 5′-TGATAGACATTAGCCAGGAG-3′; for human β-actin, the sense primer was 5′-ATCGTGCGTGACATTAAGGAG-3′ and the antisense primer was 5′-GGAAGGAAGGCTGGAAGAGT-3′. The data were normalized to the level of β-actin mRNA in each individual sample. The 2^−ΔΔCT method was used to calculate relative expression changes.

Cells were collected by centrifugation, and total RNA was extracted using the Ultrapure RNA Kit (CoWin Biotech Co. Ltd., Beijing, China). Then, RNA was reverse transcribed into cDNA using the HiFiScript cDNA Synthesis Kit (CoWin Biotech Co. Ltd., Beijing, China). The reaction conditions were as follows: 42°C for 15 minutes and 85°C for 5 minutes. The SYBR Premix Ex Taq II kit was used for qRT-PCR. The reaction conditions were as follows: 95°C for 10 seconds, 40 cycles of 95°C for 5 seconds, and 60°C for 25 seconds. The primers were as follows: UPK3A-F 5′-TCGACTCAGTCACCCCATACT-3′, UPK3A-R 5′-GAACTCCCCATGTCCACGAG-3′; p53-F 5′-TCAACAAGATGTTTTGCCAACTG-3′, p53-R 5′-ATGTGCTGTGACTGCTTGTAGATG-3′; KLF4-F 5′-CCACCTTCTTCACCCCTAGA-3′, KLF4-R 5′-AAGGTTTCTCACCTGTGTGG-3′; ZMAT3-F 5′-TCCTTCCTGTCTTGCAGGCATTT-3′, ZMAT3-R 5′-GGGAAGCCTGGGGCATAATC-3′; MDM2-F 5′-TCAGGTGATTGGTTGGATCAGG-3′, MDM2-R 5′-AGTGCATTTCCAATAGTCCTCA-3′; SP1-F 5′-CCACCATGAGCGACCAAGAT-3′, SP1-R 5′-TGAAAAGGCACCACCACCAT-3′; GADPH-F 5′-GGATTTGGTCGTATTGGGCG-3′; and GADPH-R 5′-TCCCGTTCTCAGCCATGTAGT-3′. GADPH was used as the internal reference gene. The data obtained were analyzed by the 2^−ΔΔCt method. Each experiment was performed in triplicate.

Twelve samples in three biological replicates were used to extract RNA using an Ultrapure RNA Kit (CoWin Biotech Co., China) according to the manufacturer’s instructions. The RNA was treated with RNase-free DNase I (Takara, Japan) to remove any possible DNA. The integrity was then checked by gel electrophoresis and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The concentrations of total RNA were determined using a NanoDrop 8000 spectrophotometer (NanoDrop, Wilmington, DE). Total RNA with a RIN value ≥7.3 and a 28S:18S ratio ≥ 1.2 was subjected to RNA-seq analysis by Annoroad Gene Technology Co., Ltd. (Beijing, China).

Directed by the manufacturer’s protocols, we extracted total RNA from tissues or cells using Ultrapure RNA Kit (CoWin Biosciences, China). Then the concentration was measured by a multi-wavelength microplate reader Tecan’s Infinite M200 Pro (Thermo Fisher Scientific, USA). Afterwards, PrimeScript™ RT Master Mix (Takara, Japan) was applied to transform the RNA solution into cDNA solution. The qPCR conditions were seen in the manufacturer’s protocols. GAPDH was considered as an endogenous control. All qRT-PCR assays in the paper were conducted in triplicate. TSINGKE provided us with the forward or reverse primers for CENPA and GAPDH. The primer sequences used for qPCR were: CENPA: 5′-GTG TGG ACT TCA ATT GGC AAG-3′ (forward) and 5′-TGC ACA TCC TTT GGG AAG AG-3′(reverse); CTNNB1: 5′-AAA GCG GCT GTT AGT CAC TGG-3′ (forward) and 5′-CGA GTC ATT GCA TAC TGT CCA T-3′(reverse); GAPDH: 5′-CGT GGA AGG ACT CAT GAC CA-3′ (forward) and 5′-GCC ATC ACG CCA CAG TTT C-3′ (reverse).

Quantitative real-time transcription-PCR (qRT-PCR) was used to detect the messenger RNA (mRNA) expression of the LPCAT1 gene. Total RNA was extracted using the Ultrapure RNA kit (CoWin Biosciences, Beijing, China); then, reverse transcription was conducted using the HiScript® II Q RT SuperMix for qPCR (Vazyme, Nanjing, China). qRT-PCR was performed with 2× SYBR Green PCR Master Mix (Lifeint, Xiamen, China) on a real-time fluorescent quantitative PCR meter (CFX Connect; Bio-Rad Laboratories, Hercules, CA, USA). The β-actin gene was used as an internal reference gene, and the data were analyzed using the 2^-ΔΔct method. PCR primers were ordered from Beyotime (Shanghai, China). All experiments were conducted according to the manufacturer's protocols. Experiments were performed at least in triplicate.
The PCR primer sequences used are as follows: LPCAT1 (F) 5'-CCCTGTGACCATGACGATGT-3, (R) 5'-TGAATGCACCAGGTTTGAAGGT-3' and β-actin (F) 5'-TGGCACCCAGCACAATGAA-3', (R) 5'-CTAAGTCATAGTCCGCCTAGAAGCA-3'.