Hcasm cells

To measure the expression level of lncRNAs and their neighboring protein-coding genes during differentiation of smooth muscle cells, we cultured HCASM cells (Gibco) according to the manufacturer’s protocol. These cells were maintained in Medium 231 (Gibco) supplemented with Smooth Muscle Growth Supplement (Gibco), and harvested for the sample of synthetic phenotype. To obtain the sample of contractile or differentiated phenotype, HCASM cells stabilized in the growth media were cultured in Medium 231 supplemented with Smooth Muscle Differentiation Supplement (Gibco) for three days, and then collected. We used TRIzol Reagent (Invitrogen) to extract total RNA, performed reverse transcription using RevertAid Reverse Transcriptase (Thermo Scientific) with Random Hexamers (Invitrogen). Quantitative real time polymerase chain reaction (PCR) was performed using SYBR Green PCR Master Mix (Applied biosystems) in Rotor-Gene Q (Qiagen). The primer sequences for PCR were included in the supplementary table. The differentiation of these cells was confirmed by measuring the level of Calponin 1 (CNN1) and Smooth muscle protein 22-alpha (SM22α). For normalization, the expression level of Glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used.