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Explanation of the miRNA percentage difference in WaGa wt cells (Data_Ute_smallRNA_via_exceRpt_workspace_FINAL)
Regarding your question why the miRNA percentage of the first sample, nf774, in the group “WaGa wt cells” is totally different from the other two samples, nf961 and nf962, which show 85.7% and 83.2% miRNA in exceRpt_DiagnosticPlots_WaGa.pdf, I think the drastic difference is due to a batch effect.
More specifically, nf774 was generated in an older sequencing/library preparation batch, whereas nf961 and nf962 were generated in a later optimized batch. Between these batches, the small RNA enrichment/size-selection strategy was different. The older nf774 library likely had less efficient small RNA enrichment and/or stronger adapter/junk read contamination, while nf961 and nf962 benefited from the optimized protocol and therefore show high miRNA percentages. Thus, this difference is most likely technical rather than biological.
Please find the updated sample summary and batch information below.
1. Complete Sample Table
| Sample ID | Cell Line / Type (PDF Label) | Project Batch (Sequencing Run ID) |
|---|---|---|
| Wild-Type Cells | ||
| nf961 | WaGa wt cells | 250411_VH00358_135_AAGKGLHM5 |
| nf962 | WaGa wt cells | 250411_VH00358_135_AAGKGLHM5 |
| nf774 | WaGa wt cells | 220617_NB501882_0371_AH7572BGXM_smallRNA_Ute_newDemulti |
| nf780 | MKL-1 wt cells | 220617_NB501882_0371_AH7572BGXM_smallRNA_Ute_newDemulti |
| nf796 | MKL-1 wt cells | 221216_NB501882_0404_AHLVNMBGXM_smallRNA_Ute_newDemulti |
| nf797 | MKL-1 wt cells | 221216_NB501882_0404_AHLVNMBGXM_smallRNA_Ute_newDemulti |
| WaGa EV Samples | ||
| nf657 (Excluded) | WaGa wt EV | 210817_NB501882_0294_AHW5Y2BGXJ_smallRNA_Ute_newDemulti |
| nf930, nf935 | WaGa wt EV | 231016_NB501882_0435_AHG7HMBGXV |
| nf931, nf936 | WaGa sT DMSO EV | 231016_NB501882_0435_AHG7HMBGXV |
| nf971 | WaGa sT DMSO EV | 250411_VH00358_135_AAGKGLHM5 |
| nf932, nf937 | WaGa sT Dox EV | 231016_NB501882_0435_AHG7HMBGXV |
| nf972 | WaGa sT Dox EV | 250411_VH00358_135_AAGKGLHM5 |
| nf933, nf938 | WaGa scr DMSO EV | 231016_NB501882_0435_AHG7HMBGXV |
| nf973 | WaGa scr DMSO EV | 250411_VH00358_135_AAGKGLHM5 |
| nf934, nf939 | WaGa scr Dox EV | 231016_NB501882_0435_AHG7HMBGXV |
| nf974 | WaGa scr Dox EV | 250411_VH00358_135_AAGKGLHM5 |
| MKL-1 EV Samples | ||
| nf655 (Excluded) | MKL-1 wt EV | 210817_NB501882_0294_AHW5Y2BGXJ_smallRNA_Ute_newDemulti |
| 2404, 2608 | MKL-1 wt EV | 20260506_AV243904_0073_A |
| 2608, 2701, 2802 | MKL-1 sT DMSO EV | 20260506_AV243904_0073_A |
| 2608, 2701, 2802 | MKL-1 sT Dox EV | 20260506_AV243904_0073_A |
| 2608, 2701, 2802 | MKL-1 scr DMSO EV | 20260506_AV243904_0073_A |
| 2608, 2701, 2802 | MKL-1 scr Dox EV | 20260506_AV243904_0073_A |
2. Batch Origins of the 6 wt Cell Samples
The 6 wild-type cell samples originate from 3 distinct sequencing runs:
-
220617_NB501882_0371_AH7572BGXM_smallRNA_Ute_newDemulti— June 2022- nf774, WaGa wt cells
- nf780, MKL-1 wt cells
-
221216_NB501882_0404_AHLVNMBGXM_smallRNA_Ute_newDemulti— December 2022- nf796, MKL-1 wt cells
- nf797, MKL-1 wt cells
-
250411_VH00358_135_AAGKGLHM5— April 2025- nf961, WaGa wt cells
- nf962, WaGa wt cells
In summary, nf774 comes from a different and earlier batch than nf961/nf962, which supports the interpretation that the large difference in miRNA percentage is mainly caused by batch effects and differences in library preparation/small RNA enrichment strategy.