GSM8383586: SF_X_2; Astyanax mexicanus; RNA-Seq
To extract RNA, 1 ml of TRIzol was added to each sample. Samples were homogenized, and then 200μl chloroform was added to each tube, followed by vigorous shaking. Samples were incubated on ice for 15 min, then phase separated by centrifuging at 12,000 g for 15 min at 4°C. The resulting aqueous phase of the liquid was transferred to a fresh 1.5 ml tube, and then RNA was precipitated out by mixing with 0.5 ml isopropanol. Samples were incubated on ice for 10 min, then centrifuged at 12,000 g for 10 min at 4°C. The supernatant was removed by pouring and lightly shaking the tube, then the resulting pellet was washed by the addition of 1 ml 70% EtOH while vigorously flicking the tube. The samples were then centrifuged at 7500 g for 10 min at 4°C. The supernatant was removed by pouring and lightly shaking the tube, and the samples were air dried upside down for 10 min. Finally, the pellet was redissolved in 100ul of RNAse-free H 2 O. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.