title: GSM9167516: human_hela_cell_rna_1; Homo sapiens; RNA-Seq
library_name: GSM9167516
library_construction_protocol: Skop Lab - Cells were harvested when 80% confluent with Trypsin or Versene, counted, washed with 2 mL PBS and cell pellets used for RNA extraction using the Qiagen RNeasy mini kit. RNA was eluted in RNase free water. MBRs were isolated by ultracentrifugation of cleared culture media. Cells were cultured until 80% confluent. Culture media was collected and centrifuged at 1,000×g for 10 min to clear cell debris. Cleared culture media was transferred to ultracentrifuge tubes and centrifuged at 10,000×g for 30 min at 4°C. MBR pellets were washed with 2mL sterile PBS, centrifuged at 10,000×g for 30 min at 4°C and MBR pellets used for RNA extraction using the Qiagen RNeasy mini kit. RNA was eluted in RNase free water. Iwasaki Lab - Cells were directly lysed in lysis buffer on culture dishes. Purification of desthiobiotinylated ribosomes and RNAs were performed. For ribosome profiling, RNase I treatment, ribosome pelleting, size selection, preadenylated linker ligation, rRNA depeletion, reverse transcription and PCR amplification. Skop Lab - For RNA-seq, libraries were prepared by Azenta Life Sciences using standard protocols to assess integrity, remove contaminants, and deplete ribosomal RNA. For RNA sequencing library preparation, the NEBNext Ultra II RNA Library Preparation Kit for Illumina was used according to the manufacturer's instructions. Detailed steps and reagents are available from Azenta documentation upon request. Iwasaki Lab - For Ribo-Seq, lysates were used for Thor‑Ribo‑Seq library preparation (Mito et al. 2023).