title: GSM8208479: RPF Denr shRNA2 replicate 3; Mus musculus; OTHER
library_construction_protocol: One 15 cm dish of confluent NIH3T3 cells was used per replicate. Cells were immediately harvested and flash frozen. For the generation of ribosome-protected fragments, cell lysates were treated with 5 units RNase I (ART-Seq) per OD260 for 45 min at RT, and monosomes were purified in MicroSpin S-400 columns (GE Healthcare) . RNA was extracted using miRNeasy MiniKit (Qiagen). RPF-seq and RNA-seq libraries were prepared following previously reported protocols (Arpat et al., 2020; PMID 32703885 and McGlincy and Ingolia, 2017; PMID 28579404) with minor modifications. For both RPF- and RNA-seq, 2µg of RNA was used for rRNA depletion following the RiboCop rRNA Depletion Kit V1.2 instructions from Lexogen (Cat. No. 037.24). Libraries were amplified using 10 to 14 PCR cycles, and sequenced (HiSeq2500 platform).