GSM3896450: gata2a- runx1+; Danio rerio; RNA-Seq
Total RNAs were extracted from sorted cells using RNeasy Mini Kit (QIAGEN). Cells were directly sorted in a lysis buffer containing 1 μg/mL of polyinosinic-polycytidylic acid, and total RNA was extracted using RNeasy Mini Kit. Reverse transcription (RT) was performed using Super Script III (Thermo Fisher Scientific) and an RT primer, which contains oligo-dT, T7 promoter, and PCR target region sequences. After digestion of remaining RT primers by exonuclease I (Takara), a poly-A tail was added to the 3' ends of the first-strand cDNAs using terminal transferase (Sigma). The second-strand DNA was then synthesized using MightyAmp DNA polymerase (Thermo Fisher Scientific) and a tagging primer, which contains oligo-dT and PCR target region sequences. PCR amplification was performed using a suppression primer, which allow to amplify small-size DNA that contains complementary sequences at both ends of the template DNA. The amplified double-strand cDNA was purified using QIAquick PCR Purification Kit (Qiagen). Library preparation was performed using Nextera XT DNA Library Preparation Kit (illumina).