GSM8574571: E_C5_P0; Homo sapiens; RNA-Seq
Total RNA (N=138) was isolated from uncultured (P0) and in vitro 48-hour-treated cells (Supplementary Table SIII) following established protocols from the Total RNA Purification Micro Kit (Norgen Biotek) and RNA integrity was assessed using the Agilent RNA 6000 Pico Kit and the Agilent 5200 Bioanalyzer System (Agilent Technologies Inc). Approximately 1.5-2 ng of total RNA per sample was used to generate libraries for RNA sequencing, employing the SMARTer® Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (Takara Bio Inc.). The library preparation protocol recommended by the kit was followed, with a modification to reduce reagent volumes to 1/5 of the recommended amounts Approximately 1 µL from each library was pooled and the pooled library underwent purification using the DNA Clean & Concentrator-5™ kit (Zymo Research) and the average library size was determined using the Agilent 5200 Bioanalyzer System and the Agilent DNF-474 HS NGS Fragment Kit (Agilent Technologies Inc). For quality control and normalization of all RNA libraries, the purified pooled library underwent preliminary sequencing using the Illumina NextSeq 550 System (Illumina, Inc.) for 75 cycles. The pooled library was first diluted to 4 nM in nuclease-free water and was denatured and diluted following the standard normalization method for the NextSeq 500/550 High Output v2 (75 Cycles) sequencing reagent kit (Illumina, Inc.). The final loading concentration was 1.6 pM of the denatured pooled libraries with 1% PhiX control spike-in (sequencing control).