Ficoll hypaque density gradient method

Blood samples were collected in the morning from MI patients (24-36 h after the disease onset) and CTRLs. PBMC were isolated using Ficoll-Hypaque density gradient method (Sigma-Aldrich, St. Louis, MO, USA) within 3 h of sampling. Total RNA including small RNA was extracted using miRNeasy Mini Kit (Qiagen, Hildren, Germany) following the manufacturer’s instructions. The RNA quantity was measured using the NanoDropTM spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA); the RNA integrity was assessed by QIAxcel Advanced System (Qiagen, Hilden, Germany). Samples with RNA integrity number (RIN) value above eight were included in subsequent experiments.

PBMC were isolated using Ficoll-Hypaque density gradient method (Sigma-Aldrich, St. Louis, MO, USA) within 3 h of sampling. RNA was isolated using miRNeasy Mini Kit (Qiagen, Hilden, Germany). The total RNA concentration was measured with a NanoDrop-2000 spectrophotometer and the RNA integrity was assessed by QIAxcel Advanced System (Qiagen, Hilden, Germany). Samples with a RNA integrity number (RIN) value above eight were included in subsequent experiments.

Mononuclear cells were isolated by ficoll hypaque density gradient method(density 1.077 g/ml, Sigma Aldrich, St. Louis MO).In some experiments MNCs were directly used for freezing while for the expansion procedure CD34⁺ cells were isolated by positive selection method using Dynal beads as per manufacturer’s instructions (Dynabeads M-450 CD34; Dynal, ASA, Oslo, Norway).The purity of CD34⁺cells after isolation was checked by flow-cytomter as depicted in S1A Fig it was found to be 80–90%.The 10% contaminants are MNCs from cord blood. Since, these are differentiated cells they do not proliferate further and die off during the culture period leaving behind proliferative HSCs and progenitors or differentiated CD34^- cells. Moreover we have consistently used the same CD34⁺gating for all the experiments thus minimizing the probability of intra experimental variations.