title: GSM5171826: m5B1_RP_S3; Drosophila melanogaster; OTHER
library_construction_protocol: RNA was extracted using TRIzol (Thermo Fisher Scientific) followed the manufacture protocol. For ribosome footprint libraries, 28-34mer footprints were selected on a 15% TBE-Urea gel. Dephosphorylation of the footprints were performed with shrimp alkaline phosphatase (rSAP, NEB) and ligated with 3' preadenylated adapter (rAppTGGAATTCTCGGGTGCCAAGG) by T4 RNA ligase 2, truncated (NEB). 49-55mer of ligated products were selected on a 10% TBE-Urea gel. Reverse transcription was performed with SuperScript III (company) using a primer (/5phos/GATCGTCGGACTGTAGAACTCTGAACGTGTAGATCTCGGTGGTCGC/isp18/ CACTCA/isp18/CCTTGGCACCCGAGAATTCCA) following rRNA depletion. Samples are circularized by two sequential reactions with CircLigase (Epicentre) and amplified by 9-12 PCR cycles with primers compatible with Illumina flow cells.