4200 tapestation with the high sensitivity rna screentape kit

hCOs were treated with nifedipine or DMSO (vehicle control) for 8 hours (n = 5 hCOs per condition). hCOs were washed with PBS, lysed, and stored at −80 degrees. RNA extraction was conducted using the NucleoMag 96 RNA kit (Macherey-Nagel) on the EpMotion 5075t. RNA was quantified using the Qubit RNA High Sensitivity Assay (Invitrogen). RNA integrity was assessed using the 4200 TapeStation with the High Sensitivity RNA ScreenTape kit (Agilent). Samples were normalized to ≤100 ng RNA using nuclease free water in a total of 25μl. Poly(A)-enriched RNA libraries were prepared using the Illumina Stranded mRNA Prep, Ligation (96 Samples), and the IDT for Illumina RNA UD Indexes Set A (Integrated DNA Technologies). Libraries were quantified using the Quant-iT dsDNA broad range (BR) assay kit (Invitrogen). The quality of libraries was assessed using the 4200 TapeStation with the D1000 ScreenTape kit (Agilent). Single-end 100bp sequencing was conducted on the NovaSeq 6000 (Illumina).

RNA extraction of pooled up to 10 hCO was performed using RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions. RNA was quantified using the Qubit 4.0 Fluorometer and Qubit RNA High Sensitivity Assay (Invitrogen). The 4200 TapeStation with the high sensitivity RNA ScreenTape kit (Agilent) was used to analyze the integrity of the RNA, giving an RNA Integrity Number (RIN) for each sample. Samples were normalized to <100 ng RNA in 25 μl nuclease free water. Poly(A)-enriched RNA libraries were prepared using the Illumina Stranded mRNA Prep, Ligation (96 samples), and the IDT for Illumina RNA UD Indexes Set B (UDP0177-UDP019) (Integrated DNA Technologies). Libraries were quantified using the Qubit DNA High Sensitivity Assay (Invitrogen). The quality of libraries was assessed using the 4200 TapeStation with the D1000 ScreenTape kit (Agilent). Single-end 75 bp sequencing was conducted on the NextSeq550 (Illumina). All 16 samples, samples from both experiments, were processed for RNA extraction, library preparation, and sequencing in one batch.
Extracted RNA samples and library preparation were of good quality, as shown by the Qubit reading above 1 ng/μl, RIN scores above 7 for each sample, and clear peak readings from TapeStation (Fig. 4 and Table 2 in the supplementary material).