GSM9376947: Calu3, biological replicate 3, CA, 6 h; Homo sapiens; RNA-Seq
At each collection time point, cells were treated with trypsin 0.25%, centrifuged (300g 5 min), and stored in RNAlater buffer. After the three biological replicates were collected, the total number of samples was randomly divided into groups for RNA extraction to avoid the batch effect. Before the lysis step, RNAlater buffer was removed by centrifuging samples at 5000g 15 min. Samples were lysed with RLT buffer and homogenized through QIA shredder columns (cat#79656). RNA extraction was performed with RNeasy Protect Kit (cat #74624) while incubating with RNase-free DNase I (cat #79254) treatment to eliminate DNA contamination. Samples were stored at -80°C until quantification and quality assessment using the NanoDrop One and Agilent 2100 Bioanalyzer, respectively. Subsequently, 80% of the samples tested were found to have a concentration higher than 100 ng/µL, and all of them had an RNA Integrity Number (RIN) between 8.8 and 10. RNA libraries for RNA-seq were prepared using Illumina's TruSeq mRNA Stranded kit following manufacturer's protocol.