Extended Data Fig. 3: Characterization of the BSJ-gRNA library targeting 762 circRNAs.
From: Screening for functional circular RNAs using the CRISPR–Cas13 system
a, Calculation of circRNA copy numbers (using circPOLR2A as an example) in H9, HT29, HeLa and 293FT cells. According to the FPBcirc of circPOLR2A and other circRNAs in H9, HT29, HeLa and 293FT cells, and the six copies of circPOLR2A per HeLa cell17, we calculated the copy number of all circRNAs, respectively. Copies of circPOLR2A per cell were listed. b, Schematic of circRNA copy number calculation in H9, HT29, HeLa and 293FT cells. c, Representation of 762 candidate circRNAs designed with BSJ-gRNAs in the library in different cell lines. d, Matched length distribution of 762 candidate circRNAs (solid line) and total circRNAs (dashed line). Density curve and pie chart show that more than 80% of 762 candidate circRNAs and total circRNAs are less than 1,000nt. e, Cumulative distribution of the number of reads per gRNA of constructed libraries. The red line indicates that less than 0.2% of gRNAs are covered by less than 100 reads. f, WB confirmed the stable expression of Flag-RfxCas13d in HT29, 293FT and HeLa cells used for screening. Results are representative of two independent experiments. g, KD efficiency of circRNAs remained unchanged in 30 days. RfxCas13d/BSJ-gRNA infected HT29, 293FT and HeLa cells were collected at a series of timepoints (day 1, 10, 20, 30). KD efficiency of different circRNAs was detected by qRT-PCR and were normalized to ACTB. Means ± s.d. were from three independent experiments. h, Pearson correlation coefficient (PCC) between replicates (rep) of D1 and D30 samples in HT29, 293FT and HeLa cells. Two biologically independent experiments were performed at D1 and D30 in each cell line. i, Scatter plot of fold change of paired controls and circRNA BSJ-gRNAs between D1 and D30 samples in HT29, 293FT and HeLa cells. The grey dashed lines indicate 2 or 0.5 fold change, respectively.
