GSM7917325: HEK293T (RNA-Seq), STM2457, 6h, rep4; Homo sapiens; RNA-Seq
RNA for preparation of RNA-seq library was isolated from crude cell lysate using Direct-zol RNA Miniprep kit. Possible residual DNA contamination was removed by TURBO DNase digestion. RNA was further purified using RNA Clean and Concentrator kit. Ribosome profiling was performed in HEK293T cells following a modified protocol based on McGlincy and Ingolia (PMID: 22836135) and Buchbender et al. (PMID: 31610236 ). Briefly, the lysate was treated with RNase I and nuclease digestion was stopped by adding SUPERase In RNase inhibitor. Ribosomes were isolated by sucrose cushion and centrifugation with a TLA100.3 rotor at 100.000 rpm, 4°C for 1 h. RNA was purified from the resuspended ribosomal pellet using the Direct-zol kit according to the manufacturer's instructions for purifying total RNA including small RNAs. The eluted RNA was precipitated with ethanol. For the purification of ribosome-protected fragments, RNA samples were run on a 15% polyacrylamide TBE-urea gel for 65 min at 200 V next to synthesized marker oligonucleotides of defined length (26 nt and 34 nt). The gel regions between 26–34 nt were excised for each sample and RNA was eluted in the extraction followed by phenol-chloroform extraction and RNA precipitation with isopropanol. T4 polynucleotide kinase was used to remove 2'-3' cyclic phosphate from ribosomal footprints. After 3'-linker ligation, unligated 3'-linker was removed from the reaction by the addition of 0.5 µl yeast 5'-deadenylase and 0.5 µl RecJ exonuclease. The RNA was purified with Oligo Clean and Concentrator kit according to the manufacturer's instructions. RNA libraries for RNA-seq were prepared using TruSeq Strand-Specific mRNA Library Prep with PolyA-Selection. For Ribo-seq, reverse transcription was performed at the following conditions: 25°C for 5 min, 55°C for 60 min, 80°C for 5 min, and held at 4°C. To remove RNA, alkaline hydrolysis was performed. The second adapter ligation to the 3' end of the cDNA was performed as described in Buchbender et al. (PMID: 31610236 ). Briefly, after reverse transcription, cDNA clean-up was performed using MyONE Silane beads. Next, a second linker ligation was performed using different L##clip2.0 oligoes for each sample. 5'-linker ligated cDNA was cleaned up using MyONE Silane beads. Different from the published protocol (ref), we omitted PCR preamplification, followed by the first ProNex size selection before preparative PCR amplification, and instead performed just one PCR amplification step with an optimized number of cycles. After PCR, different samples were multiplexed in equal molarities. The library was then purified, and size-selected on ProNex magnetic beads. The library was sequenced on an Illumina NextSeq 500 with more than 40 million reads