GSM4483526: oe3.ribo: mimic miR-1271-5p 3; Homo sapiens; OTHER
Cells were exposed to cycloheximide (0.1mg/mL) prior to detergent lysis and scraping as per the Illumina TruSeq Ribo Profile kit manual. Ribosome profiling was conducted using the Illumina TruSeq Ribo Profile Kit (H/M/R) according to the manufacturer's instructions, with minor amendments. To inhibit translational activity, transfected cells were treated with culture medium supplemented with 0.1mg/mL cycloheximide (CHX) for 1 min. Cells were subsequently washed with ice-cold CHX-supplemented PBS, then harvested on ice via the addition of 1mL mammalian lysis buffer (containing 200µL 5x mammalian polysome buffer, 2µL CHX and 10 units (U) DNase I) and extensive scraping. Ribosome foot-printing was performed by adding 90U RNase I to 300µL clarified lysate and incubating samples at room temperature for 45 min, after which reactions were stopped with 15µL SUPERase inhibitor (Invitrogen). Ribosome-protected RNA fragments (RPFs) were then enriched using MicroSpin S-400 columns (GE Life Sciences), prior to ribosomal RNA depletion, RPF size selection, end repair, adapter ligation, reverse transcription and PAGE purification, conducted according to the manufacturer's instructions. RPF cDNA libraries were subsequently produced with 12 PCR cycles, following which adapter dimers were removed via 8% native polyacrylamide gel prior to library normalization and pooling. RPF libraries were then sequenced using the Illumina NextSeq 500 system, with a total of 76 single-end cycles performed.
GEO Accession
GSM4483526