Nanodrop bioanalyzer

Total RNA was isolated from MDA-MB-231 breast cancer cells expressing parental (Scrambled), increased (Up), decreased (Down, CRISPR 2–12), and knockout (CRISPR 2–19, CRISPR 5–50) levels of NAT1 using the RNeasy^® Mini Kit (Qiagen Sciences, Germantown, Maryland) according to manufacturer’s instructions. RNA quality was evaluated and concentrations were measured in each sample using a NanoDrop Bioanalyzer (Thermo Fischer Scientific).

The human nucleus pulposus cells used in this experiment were purchased from ScienCell. After defrosting, cells were inoculated into tissue culture dishes and cultured in DMEM complete medium supplemented with 1% penicillin-streptavidin, 1% L-glutamine, and 10% fetal calf serum in a 37°C humidified incubator. The cells were subcultured at a dilution of 1 : 3 when they reached 90% confluency. To test the induction of COX2 by IL-1β, cells at P3 were subjected to the addition of IL1β at the final concentration of 0, 5, 10, and 15 ng/ml for 24 hr. After 24 hr, the cells were harvested. RNA was isolated by using Trizol and assessed by using a Nanodrop bioanalyzer (Thermo Scientific, US). Reverse transcription of RNA to cDNA was done with an RNA to cDNA kit (Tsingke, PRC). Quantitative real-time PCR (qRT-PCR) of the expression of COX2 was performed on a StepOnePlus system (Applied Biosystems, Life technologies, US) using SYBR green real-time PCR master mixes (Tsingke, PRC). GAPDH was tested as an endogenous control. The relative quantification was achieved by the comparative CT method.

Total RNA was extracted from the thousands of synchronized worms using Trizol (Invitrogen, Carlsbad, USA) according to the manufacturing instructions. RNA was qualified and quantified using a Agilent 2100 and NanoDrop bioanalyzer (Thermo Fisher Scientific, USA). Qualified lncRNA libraries were sequenced via pair-end sequencing on the Hiseq xten platform [34 (link)]. Qualified small RNA libraries via pair-end sequencing on the BGISEQ-500 platform (BGI-Shenzhen, China) [35 (link)].