title: GSM9160119: EPN24-Rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Cell supernatant was clariﬁed by centrifugation at 3,000 g for 5 minutes, then filtrated through a cellulose acetate ﬁlter with 0.45 μm pore size (Corning). 200 μl of supernatant was mixed with 800 μl TRIzol reagent (Magen) and 1 μl diluted External RNA Controls Consortium (ERCC,1:1000) synthetic spike-in RNAs (ThermoFisher). RNA was extracted and dissolved in 20 μl nuclease-free water. 5 μl supernatant RNA was used for library construction with modified VAHTS Universal V10 RNA-seq Library Prep Kit for Illumina (Vazyme). Typically,  5 μl RNA was mixed with 5 μl 2×Prime buffer (Vazyme) and 0.7 μl FastSelect reagent (Vazyme) for fragmentation at 85℃ for 6 min. Then 8 μl fragmentated RNA was used for first and secondary strand cDNA synthesis, followed by adapter ligation and PCR amplification 17 cycles according to the manufacture's instruction manual. The DNA library was purified with VAHTS DNA Clean Beads (Vazyme) and dissolved in 20 μl nuclease-free water. The 150-nt paired-end sequencing was performed on  DNBSEQ-T7 platform (GenePlus, China).