GSM2975549: S2 cell ribosomal footprinting AGO1-dsRNA-treated replicate 2; Drosophila melanogaster; RNA-Seq
Ribosomal footprinting was carried out as described in (Calviello et al. 2016), with minor changes. In brief, cells were rinsed from the plate and transferred to a 50 ml falcon tube, preloaded with Cycloheximide (SIGMA #C4859) for a final concentration of 0.1 mg/ml. Cells were spun down at 200g for 5 min at 4°C and washed once with cold PBS supplemented with 0.1 mg/ml CHX. After aspiration, the cell pellet was snap-frozen in liquid nitrogen. The subsequent lysis, footprinting and recovery of ribosome-protected fragments were carried out as described in (Calviello et al. 2016). Ribosomal footprinting libraries were then generated using the NEXTflex Small RNA Library Prep Kit v3 (Bioo Scientific #5132) using rRNA-depleted (RiboZero; Illumina #MRZG12324) and T4 PNK (NEB #M0201) treated RNA as input. By pilot-PCR the minimal PCR cycle number for library amplification was determined (AGO1 samples 14-15 cycles). Amplicons were gel-purified prior to sequencing a multiplexed pool on one flow cell (NextSeq500 high output mode, 75 cycles single-end.)
GEO
GSM2975549