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Experiment Summary

This report summarizes a miR-eCLIP experiment performed on 7 samples: Sample1_IP, Sample2_IP, Sample3_IP, Sample4_IP, Sample5_IP, Sample6_IP and Sample7_IP. For miR-eCLIP experiments, the standard eCLIP protocol [PMID 27018577] was modified to enable chimeric ligation of miRNA and mRNA according to bioRxiv preprint by Manakov et al, 2022 [10.1101/2022.02.13.480296]. While plated, approximately 20 x 106 cells were UV crosslinked at 400 mJoules/cm2 with 254 nm radiation, pelleted, snap frozen on ice dry and stored until use at -80°C. Cell pellets were then lysed with 1 mL of eCLIP lysis mix and sonicated (QSonica Q800R2) for 5 minutes, 30 seconds on / 30 seconds off with an energy setting of 75% amplitude, followed by digestion with RNase-I (Ambion). A primary mouse monoclonal AGO2/EIF2C2 antibody (sc-53521, Santa Cruz Biotechnology) was incubated for 1 hour with magnetic beads pre-coupled to the secondary antibody (M-280 Sheep Anti-Mouse IgG Dynabeads, Thermo Fisher 11202D) and added to the homogenized lysate for overnight immunoprecipitated at 4°C. Following overnight IP, 2% of the sample was taken as the paired size-matched input with the remainder magnetically separated and washed with eCLIP high stringency wash buffers. Chimeric ligation was then performed on-bead at room temperature for 1 hour with T4 RNA ligase (NEB). IP samples were then dephosphorylated with alkaline phosphatase (FastAP, Thermo Fisher) and T4 PNK (NEB) and an RNA adapter was ligated to the 3′ ends. IP and input samples were cut from the membrane at the AGO2 protein band size to 75 kDa above. Western blot was visualized using anti-AGO2 primary antibody (50683-RP02, SinoBiological) at a 1:2000 dilution, with TrueBlot anti-rabbit secondary antibody (18-8816-31, Rockland) at 1:6000 dilution. RNA adapter ligation, IP-western, reverse transcription, DNA adapter ligation, and PCR amplification were performed as previously described. Sequencing was performed as SE122 on the NextSeq 2000 platform.

After sequencing, samples were processed with Eclipsebio's proprietary analysis pipeline (v1). UMIs were pruned from read sequences using umi_tools (v1.1.1). Next, 3' adapters were trimmed from reads using cutadapt (v3.2). Reads were then mapped to a custom database of repetitive elements and rRNA sequences. All non-repeat mapped reads were mapped to the genome (UCSC version GRCh38/hg38) using STAR (v2.7.7a). PCR duplicates were removed using umi_tools (v1.1.1). AGO2 eCLIP peaks were identified within eCLIP samples using the peak caller CLIPper (v2.0.1). For each peak, IP versus input fold enrichments and p-values were calculated. miRNAs from miRBase (v22.1) were "reverse mapped" to any reads that did not map to repetitive elements or the genome using bowtie (v1.2.3). The miRNA portion of each read was then trimmed, and the remainder of the read was mapped to the genome using STAR (v2.7.7a). PCR duplicates were resolved using umi_tools (v1.1.1), and miRNA target clusters were identified using CLIPper (v2.0.1). Each cluster was annotated with the names of miRNAs responsible for that target. Peaks were annotated using transcript information from GENCODE release 41 (GRCh38.p13) with the following priority hierarchy to define the final annotation of overlapping features: protein coding transcript (CDS, UTRs, intron), followed by non-coding transcripts (exon, intron).

miR-eCLIP Workflow