D1000 screentape on a 2200 tapestation

hCEC clones were plated in 6-well plates 1 day before the collection. On the second day, the cells were checked to make sure their confluency was within 70–90% and morphology was normal. Then the cells were washed twice in PBS and stored at –80°C immediately. Total RNA was isolated from each sample using PicoPure RNA Isolation kit (Life Technologies, Frederick, MD) including the on-column RNase-free DNase I treatment (QIAGEN, Hilden, Germany) following the manufacturer’s recommendations. To purify RNA for sequencing, we used the QIAGEN RNeasy Mini Kit (QIAGEN 74106). RNA concentration and integrity were assessed using a 2100 BioAnalyzer (Agilent, Santa Clara, CA). Sequencing libraries were constructed using the TruSeq Stranded Total RNA Library Prep Gold mRNA (Illumina, San Diego, CA) with an input of 250 ng and 13-cycle final amplification. Final libraries were quantified using High Sensitivity D1000 ScreenTape on a 2200 TapeStation (Agilent) and Qubit 1x dsDNA HS Assay Kit (Invitrogen, Waltham, MA). Samples were pooled equimolar with sequencing performed on an Illumina NovaSeq6000 SP 100 Cycle Flow Cell v1.5 as paired-end 50 reads.