GSM8942734: mouse LSK(Lin−Sca-1+c-Kit+) cells, Afg3l2-KO, rep1; Mus musculus; RNA-Seq
Total RNA was isolated from WT control or Afg3l2-KO LSK(Lin−Sca-1+c-Kit+) cells using FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme). mRNA was purified from total RNA by using poly-T oligo-attached magnetic beads. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase, then RNA was digested with RNase H. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and dNTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR amplification, the PCR product was purified by AMPure XP beads, and the library was finally obtained. Each group was sequenced in replicates by Illumina NovaSeq 6000 with single end 150 bp read length.