M mlv rt

Reverse transcription was performed at 37 °C for 2 h in a total volume of 10 μL reaction solution containing 3 μg RNA, 0.5 μg oligo (dT), 1X moloney murine leukemia virus reverse trancscriptase (MMLV RT) buffer, 0.5 mm each dNTP, 0.1 mm dithiothreitol and 100 U MMLV RT (Invitrogen, Carlsbad, CA, USA). To validate the RNA‐seq data, we determined the expression levels of several selected lncRNA transcripts by real‐time quantitative PCR in both day5_ybx1^+/+ and day5_ybx1^−/−, including MSTRG12630.1, MSTRG24792.1, ENSDART00000171757, MSTRG.30533.1 and MSTRG.33365.1. To study the correlation between the selected lncRNA and their targets, including duox (NADPH oxidase), noxo1a (NADPH oxidase organizer) and ybx1, we determined the mRNA expression levels of targets by real‐time quantitative PCR after targeted lncRNA morpholino microinjection. The expression levels were normalized to that of the housekeeping gene ef1a. The standard for each gene was prepared by PCR amplification of cDNA fragments with specific primers (Table 1). The real‐time quantitative PCR assay was performed on the CFX96 Real‐time PCR Detection System (Bio‐Rad, Hercules, CA, USA) and repeated twice. All values were expressed as the mean ± standard error of the mean (n = 3), and the data were analyzed by t‐test using Prism 6 on Macintosh OS X (GraphPad Software, San Diego, CA, USA).

Total RNA was extracted from cells using RNeasy kit (Qiagen, Germany). 2 × 10⁶ cells were centrifuged and washed twice with PBS. RNA was isolated from these cells as per the manufacturer's instructions. 1.5 μg of total RNA was converted to cDNA using MMLV-RT (Thermo scientific, USA) and Oligo-dT primers (NEB, USA). miRNA conversion to cDNA was carried out using stem-loop reverse transcriptase (RT) primers without dithiothreitol (DTT) and the RNA denaturation step to maintain integrity of the stem-loop primer.