Large scale rna production system t7

After linearization of plasmids, in vitro T7 RNA polymerase transcription (Large Scale RNA Production System‐T7; Promega) and Biotin RNA Labeling (RNA 3′ End Desthiobiotinylation Kit; Thermo Fisher Scientific) were used to synthesise transcripts of lincRNA‐EPS full‐length and mutant fragments. The primers are presented in Table S2. Meanwhile, protein lysates from LPS + ATP‐treated NIH3T3 cells were prepared. Then, the biotin RNAs were incubated with the lysate overnight, and a pull‐down assay was implemented in accordance with the instructions of the Pierce Magnetic RNA–Protein Pull‐Down Kit (Thermo Fisher Scientific). Finally, the retrieved proteins were detected by mass spectrometry (MS) at Shanghai Dian Xi Bio Co. Ltd.

Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher, cat. no. 20164) provides reagents to efficiently enrich RNA Binding Proteins. Sense or antisense of lnc9456 or lnc4012 were in vitro transcription using the T7 RNA transcription system (Large Scale RNA ProductionSystem-T7, Promega, cat. no. P1300) and biotin-labeled with the Pierce™ RNA 3’ End Desthiobiotinylation Kit (Thermo Scientific, cat. no. 20163). The primer sequences were: lnc9456-sense: 5’-TAATACGACTCACTATAGGGGTGACACATCTGGAGATTTTCC-3’ (forward), 5’-AGTCAGAGGATAACTTGGGGG-3’ (reverse); lnc9456-antisense: 5’-GTGACACATCTGGAGATTTTCC-3’ (forward), 5’-TAATACGACTCACTATAGGGAGTCAGAGGATAACTTGGGGG-3’ (reverse); lnc4012-sense: 5’-TAATACGACTCACTATAGGGCAGGCCAAGCTTGCTTCCTAC-3’ (forward), 5’-TCAAGCCATGAGTCAGCCTAA-3’ (reverse); lnc4012-antisense: 5’-CAGGCCAAGCTTGCTTCCTAC-3’ (forward), TAATACGACTCACTATAGGGTCAAGCCATGAGTCAGCCTAA-3’ (reverse). RNA was then purified with QIAquick PCR Purification Kit (Qiagen, cat. no. 28106). 30 μg of whole-cell protein lysates were incubated with purified biotinylated transcripts for 1 h at 4 °C with rotation, then they were eluted for mass spectrometry identification. RNA pull-down and analysis were performed by Shanghai Dianxi Biotechnology Co., Ltd.