GSM9558356: D29_A; Homo sapiens; RNA-Seq
Cells were plated on 6-well plates one day before the collection. On the second day, the cells were examined to ensure their confluency level between 70% to 90%, with normal morphological characteristics. Then the cells were washed twice with PBS and promptly stored at –80°C. Total RNA was extracted using the PicoPure RNA Isolation kit (Life Technologies, Frederick, MD), which included an on-column RNase-free DNase I treatment (QIAGEN, Hilden, Germany), following the manufacturer's recommendations. To further purify the RNA for sequencing, the QIAGEN RNeasy Mini Kit (QIAGEN, 74106) was used. The concentration and integrity of the RNA were assessed using a 2100 BioAnalyzer (Agilent, Santa Clara, CA). Sequencing libraries were constructed using the TruSeq Stranded Total RNA Library Prep Gold mRNA kit (Illumina, San Diego, CA) with an input of 250 ng RNA and a 13-cycle final amplification. The resulting libraries were quantified using High Sensitivity D1000 ScreenTape on a 2200 TapeStation (Agilent) and the Qubit 1x dsRNA HS Assay Kit (Invitrogen, Waltham, MA). The samples were equimolarity pooled, and sequencing was carried out on an Illumina NovaSeq6000 SP 100 Cycle Flow Cell v1.5 in paired-end 50 reads mode.