design: Characterisation of the AGO2 binding sites in HeLa cell line using eiCLIP
library_construction_protocol: HeLa cells were maintained in RPMI 1640 or DMEM medium, respectively, supplemented with 10% foetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin at 37°C in 5% CO2 in a humidified incubator.  AGO2-eiCLIP was performed as previously described (Paterson et al. 2022). In brief, this involved following a previously described non-isotopic iCLIP workflow (Sibley 2018) which had additional modifications to enhance speed and efficiency. This included ligation of a new Cy5.5 labelled adapter (/5Phos/A[XXXXXX]NNNAGATCGGAAGAGCACACG/3Cy55Sp/) to bound RNA with  high concentration T4 RNA ligase (New England Biolabs), use of RecJf exonuclease (New England Biolabs) to remove un-ligated adapter prior to SDS-PAGE analysis, reverse transcription with a biotinylated primer (/5BiotinTEG/CGTGTGCTCTTCCGA), exonuclease III (New England Biolabs) mediated removal of unextended RT-primer, cDNA capture with MyOne C1 streptavidin beads (ThermoFisher Scientific), 3' adapter (/5Phos/ANNNNNNNAGATCGGAAGAGCGTCGTG/3ddC/) ligation instead of intramolecular ligation, and cDNA elution with nuclease and cation free water at high temperature. For a pellet of cells obtained from a 80% confluent 150mm dish, we used 100 µl Dynabeads Protein G (Thermo Fisher Scientific) conjugated to 1.5 µg anti-AGO2 antibody (MAB253, Sigma-Aldrich/ Merck).  Sample was sequenced with single-end reads using NextSeq500.