GSM9307196: HEK293 shSTUB1 37°C control rep2; Homo sapiens; RNA-Seq
Immediately after the treatment timepoint, culture medium was aspirated and cells were lysed directly on the plate by addition of RLT buffer (QIAGEN). Total RNA was then extracted using the RNeasy Mini Kit (QIAGEN cat. no. 74104) with on-column DNase I digestion (QIAGEN RNase-Free DNase Set, cat. no. 79254) according to the manufacturer's instructions, including the optional β-mercaptoethanol step in RLT for complete denaturation. RNA concentration and purity were assessed by NanoDrop; integrity was confirmed by Agilent Bioanalyzer 2100 (RIN ≥ 8.0 for all samples). Polyadenylated mRNA was purified from 500–1000 ng total RNA and sequencing libraries were prepared using the Illumina TruSeq RNA Sample Preparation Kit v2 (cat. no. RS-122-2001 or RS-122-2002) following the manufacturer's low-throughput protocol. Libraries were amplified for 15 PCR cycles and size-selected for ~200–300 bp inserts. Final library quantity and quality were assessed by Qubit and Agilent Bioanalyzer. Indexed libraries were pooled equimolarly (6-plex per lane) and clustered using the Illumina cBot. Sequencing was performed single-end 50 cycles on an Illumina HiSeq 2500 using v3 or v4 chemistry at the University of North Carolina High-Throughput Sequencing Facility (HTSF).