Brunner et al_2026_Raw data

Fig. 2B: Vulva bursting quantification in heterochronic mutants Quantification of vulva bursting of adult animals of indicated genotypes. Animals were grown at 25°C on NG2% OP50 plates and examined at 48 h of development. N = 3 technical replicates. Related files: Fig2B_Vulva bursting quantification in heterochronic mutants.xlsx Condition Strain code Genotype WT N2 WT let-7(-) HW2569 let-7(xe150) X; xeEx365[let-7p::let-7::SL1_operon::gfp, unc-119 (+); rab-3p::mCherry; myo-2p::mCherry; myo-3p::mCherry] lin-28(0) HW2705 lin-28(xe192) I lin-28(0); let-7(-) HW2792 lin-28(xe192) I; let-7(xe150) X lin-28(0); lin-46(0) HW3336 lin-28(xe192) I; lin-46(ma385) V lin-46(0) HW3506 lin-46(ma385) V lin-28(0); lin-46(0); let-7(-) HW3677 lin-28(xe192) I; lin-46(ma385) V; let-7(xe150) X; xeEx365[let-7p::let-7::SL1_operon::gfp, unc-119 (+); rab-3p::mCherry; myo-2p::mCherry; myo-3p::mCherry] lin-46(0); let-7(-) HW3678 lin-46(ma385) V; let-7(xe150) X; xeEx365[let-7p::let-7::SL1_operon::gfp, unc-119 (+); rab-3p::mCherry; myo-2p::mCherry; myo-3p::mCherry] Fig. 3A_Fig. 6B_Fig. S7B: Alae quantification Quantification of the presence of alae in animals of indicated genotypes. Molting animals were singled and examined shortly after they exited the respective molt as indicated by ecdysis. The column "Animals singled at (h)" refers to the time after plating of synchronized L1s at which molting animals were singled. Related files: Alae_quantification_M2.xlsx, Alae_quantification_M3.xlsx, Alae_quantification_M4.xlsx Fig. 4AB_Fig. 7D_Fig. S8: Luciferase assay heterochronic mutants Luciferase assay data used to quantify the duration of larval stages of animals of the indicated genotypes. The file (Fig4AB_7D_S8_Luciferase assay.xlsx) contains three sheets: Raw data: Contains the raw values measured by the luminometer (Berthold Technologies, Centro XS3 LB 960). Luminescence was measured every 10 min for 0.5 seconds. The luminometer was placed in a temperature-controlled incubator set to 18.5°C, which translated to an effective temperature of 22.5°C experienced by the animals inside the luminometer. Plate design: Position of animals of indicated genotypes in the 384-well luminometer plate. In let-7(0), lin-46(0); let-7(0) and the triple mutant animals, only wells containing unbalanced animals were labeled and considered for the analysis. Annotated data: List of animals of indicated genotypes with the times of hatch, molt entry and exit and calculated durations of molt, intermolts and larval stages in hours. Animals excluded from the analysis are indicated by "0" in the "is_valid" column. Condition Strain code Genotype WT HW1993 xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV lin-46(0) HW3505 xeSi312 [eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV; lin-46(ma385) V let-7(0) HW2791 xeSi312 [eft-3p::luc::gfp::unc-54 3’UTR, unc-119(+)] IV; let-7(xe150) X; xeEx365 [let-7p::let-7::SL1_operon_GFP, unc-119 (+); Prab-3::mCherry; Pmyo-2::mCherry; Pmyo-3::mCherry] lin-28(0); let-7(0) HW2793 lin-28(xe192) I; xeSi312 [eft-3p::luc::gfp::unc-54 3’UTR, unc-119(+)] IV; let-7(xe150) X lin-28(0); lin-46(0) HW3504 lin-28(xe192) I; xeSi312 [eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV; lin-46(ma385) V lin-46(0); let-7(0) HW3544 xeSi312 [eft-3p::luc::gfp::unc-54 3’UTR, unc-119(+)] IV; lin-46(ma385) V; let-7(xe150) X; xeEx365 [let-7p::let-7::SL1_operon_GFP, unc-119 (+); Prab-3::mCherry; Pmyo-2::mCherry; Pmyo-3::mCherry] lin-28(0) HW2706 lin-28(xe192) I; xeSi312 [eft-3p::luc::gfp::unc-54 3’UTR, unc-119(+)] IV triple mut HW3543 lin-28(xe192) I; xeSi312 [eft-3p::luc::gfp::unc-54 3’UTR, unc-119(+)] IV; lin-46(ma385) V; let-7(xe150) X; xeEx365 [let-7p::let-7::SL1_operon_GFP, unc-119 (+); Prab-3::mCherry; Pmyo-2::mCherry; Pmyo-3::mCherry] Fig. 4D: Luciferase assay let-7(0) on cdk-1 RNAi Luciferase assay data used to quantify the duration of larval stages of animals of the indicated genotypes. The animals were fed E. coli OP50 bacteria or E. coli HT115 bacteria, either containing the insert-less L4440 parental RNAi vector (denoted “EV”) or an RNAi vector with an insert targeting the gene of interest (cdk-1 or grh-1 as a technical positive control). The file (Fig4D_Luciferase assay.xlsx) contains three sheets: Raw data: Contains the raw values measured by the luminometer (Berthold Technologies, Centro XS3 LB 960). Luminescence was measured every 10 min for 0.5 seconds. The luminometer was placed in a temperature-controlled incubator set to 18.5°C, which translated to an effective temperature of 22.5°C experienced by the animals inside the luminometer. Plate design: Position of animals of indicated genotypes in the 384-well luminometer plate. In let-7(0), lin-46(0); let-7(0) and the triple mutant animals, only wells containing unbalanced animals were labeled and considered for the analysis. Annotated data: List of animals of indicated genotypes with the times of hatch, molt entry and exit in hours. Animals excluded from the analysis are indicated by "0" in the "is_valid" column. Condition Strain code Genotype let-7(0) HW2791 xeSi312 [eft-3p::luc::gfp::unc-54 3’UTR, unc-119(+)] IV; let-7(xe150) X; xeEx365 [let-7p::let-7::SL1_operon_GFP, unc-119 (+); Prab-3::mCherry; Pmyo-2::mCherry; Pmyo-3::mCherry] triple mut HW3543 lin-28(xe192) I; xeSi312 [eft-3p::luc::gfp::unc-54 3’UTR, unc-119(+)] IV; lin-46(ma385) V; let-7(xe150) X; xeEx365 [let-7p::let-7::SL1_operon_GFP, unc-119 (+); Prab-3::mCherry; Pmyo-2::mCherry; Pmyo-3::mCherry] lin-46(0); let-7(0) HW3544 xeSi312 [eft-3p::luc::gfp::unc-54 3’UTR, unc-119(+)] IV; lin-46(ma385) V; let-7(xe150) X; xeEx365 [let-7p::let-7::SL1_operon_GFP, unc-119 (+); Prab-3::mCherry; Pmyo-2::mCherry; Pmyo-3::mCherry] Fig. 6A: Pvl phenotype quantification I Quantification of the protruding vulva (Pvl) phenotype of adult animals of indicated genotypes. Animals were grown at 20°C and examined at 52 h of development after plating synchronized L1 animals. N = 4 technical replicates. Related files: Fig6A_Pvl phenotype quantification_I.xlsx Condition Strain code Genotype wild type N2 wild type wild type* HW1792 lin-28(xe59[lin-28::flag::ha]) I lin-28(Δb)*; lin-46(++) HW3696 lin-28(xe344xe59[lin-28::flag::ha with exon1B(Δ)]) I; lin-46(xe370[5'UTR_GGAGmut]) V lin-28(Δb)*; let-7(++) HW3760 lin-28(xe344xe59[lin-28::flag::ha with exon1B(Δ)]) I; xeSi287[let-7p::let-7] V lin-28(Δb)* HW3381 lin-28(xe344xe59[lin-28::flag::ha with exon1B(Δ)]) I let-7(++) lin-46(++) HW3808 xeSi287[let-7p::let-7] lin-46(xe370[5'UTR_GGAGmut]) V lin-28(0) HW2705 lin-28(xe192) I lin-46(++) HW3531 lin-46(xe370[5'UTR_GGAGmut]) V let-7(++) HW1909 xeSi287[let-7p::let-7] V lin-28(Δb)*; let-7(++) lin-46(++) HW3810 lin-28(xe344xe59[lin-28::flag::ha with exon1B(Δ)]) I; xeSi287[let-7p::let-7] lin-46(xe370[5'UTR_GGAGmut]) V Fig. 6CD_Fig. 7E: Luciferase assay let-7 and lin-46 overexpression in lin-28(Δb) background Luciferase assay data used to quantify the duration of larval stages of animals of the indicated genotypes. The file (Fig6CD_7E_Luciferase assay.xlsx) contains three sheets: Raw data: Contains the raw values measured by the luminometer (Berthold Technologies, Centro XS3 LB 960). Luminescence was measured every 10 min for 0.5 seconds. The luminometer was placed in a temperature-controlled incubator set to 18.5°C, which translated to an effective temperature of 22.5°C experienced by the animals inside the luminometer. Plate design: Position of animals of indicated genotypes in the 384-well luminometer plate. Annotated data: List of animals of indicated genotypes with the times of hatch, molt entry and exit and calculated durations of molt, intermolts and larval stages in hours. Animals excluded from the analysis are indicated by "0" in the "is_valid" column. Condition Strain code Genotype wild type HW1993 xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV wild type* HW3692 lin-28(xe59[lin-28::flag::ha]) I; xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV lin-28(0) HW2706 lin-28(xe192) I; xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV lin-28(Δb)* HW3454 lin-28(xe344xe59[lin-28::flag::ha with del_exon1B]) I; xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV let-7(++) HW3771 xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV; xeSi287[let-7p::let-7] V lin-46(++) HW3530 xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV; lin-46(xe370) V let-7(++) lin-46(++) HW3809 xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV; xeSi287[let-7p::let-7] lin-46(xe370) V lin-28(Δb)*; let-7(++) HW3759 lin-28(xe344xe59[lin-28::flag::ha with del_exon1B]) I; xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV; xeSi287[let-7p::let-7] V lin-28(Δb)*; lin-46(++) HW3695 lin-28(xe344xe59[lin-28::flag::ha with del_exon1B]) I; xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV; lin-46(xe370) V lin-28(Δb)*; let-7(++) lin-46(++) HW3811 lin-28(xe344xe59[lin-28::flag::ha with del_exon1B]) I; xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV; xeSi287[let-7p::let-7] lin-46(xe370) V Fig. 7AC: Luciferase assay lin-28 mutant alleles Luciferase assay data used to quantify the duration of larval stages of animals of the indicated genotypes. The file (Fig7AC_Luciferase assay.xlsx) contains three sheets: Raw data: Contains the raw values measured by the luminometer (Berthold Technologies, Centro XS3 LB 960). Luminescence was measured every 10 min for 0.5 seconds. The luminometer was placed in a temperature-controlled incubator set to 18.5°C, which translated to an effective temperature of 22.5°C experienced by the animals inside the luminometer. Plate design: Position of animals of indicated genotypes in the 384-well luminometer plate. Annotated data: List of animals of indicated genotypes with the times of hatch, molt entry and exit and calculated durations of molt, intermolts and larval stages in hours. Animals excluded from the analysis are indicated by "0" in the "is_valid" column. Condition Strain code Genotype wild type HW1993 xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV lin-28(0) HW2706 lin-28(xe192) I; xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV lin-28(n719) HW2790 lin-28(n719) I; xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV Fig. 7F_Fig. S7CD: Luciferase assay let-7 and lin-46 overexpression in lin-28::large_tag background Luciferase assay data used to quantify the duration of larval stages of animals of the indicated genotypes. The file (Fig7F_S7CD_Luciferase assay.xlsx) contains three sheets: Raw data: Contains the raw values measured by the luminometer (Berthold Technologies, Centro XS3 LB 960). Luminescence was measured every 10 min for 0.5 seconds. The luminometer was placed in a temperature-controlled incubator set to 18.5°C, which translated to an effective temperature of 22.5°C experienced by the animals inside the luminometer. Plate design: Position of animals of indicated genotypes in the 384-well luminometer plate. Annotated data: List of animals of indicated genotypes with the times of hatch, molt entry and exit and calculated durations of molt, intermolts and larval stages in hours. Animals excluded from the analysis are indicated by "0" in the "is_valid" column. Condition Strain code Genotype wild type HW1993 xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV lin-28(0) HW2706 lin-28(xe192) I; xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV let-7(++) HW3771 xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV; xeSi287[let-7p::let-7] V lin-46(++) HW3530 xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV; lin-46(xe370) V let-7(++) lin-46(++) HW3809 xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV; xeSi287[let-7p::let-7] lin-46(xe370) V lin-28::large_tag HW3226 lin-28(xe197) I; xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV lin-28::large_tag; let-7(++) HW3757 lin-28(xe197) I; xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV; xeSi287[let-7p::let-7] V lin-28::large_tag; lin-46(++) HW3684 lin-28(xe197) I; xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV; lin-46(xe370) V lin-28::large_tag; let-7(++) lin-46(++) HW3882 lin-28(xe197) I; xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV; xeSi287[let-7p::let-7] lin-46(xe370) V Fig. S3: qPCR let-7 alleles validation Raw qPCR data supporting the Fig. S3. This entry contains three datasets: FigS3B_qPCR_xe150 validation_mature.xlsx: The levels of mature let-7 and sn2841 (reference gene) were measured by RT-qPCR using the TaqMan chemistry in the strains listed below at 30 and 33 hours of growth at 25°C (starting from synchronized L1 population). Only unbalanced let-7(-) animals were used for the experiment. Condition Strain code Genotype wild type N2 wild type let-7(-) HW2569 let-7(xe150) X; xeEx365[let-7p::let-7::SL1_operon::gfp, unc-119 (+); rab-3p::mCherry; myo-2p::mCherry; myo-3p::mCherry] FigS3C_qPCR_xe150 validation_primary.xlsx: The levels of primary let-7 transcript and actin mRNA (reference gene) were measured by RT-qPCR in the strains listed below at 30 and 33 hours of growth at 25°C (starting from synchronized L1 population). Only unbalanced let-7(-) animals were used for the experiment. Condition Strain code Genotype wild type N2 wild type let-7(-) HW2569 let-7(xe150) X; xeEx365[let-7p::let-7::SL1_operon::gfp, unc-119 (+); rab-3p::mCherry; myo-2p::mCherry; myo-3p::mCherry] FigS3D let-7 BS.xlsx: The levels of mature let-7 and sn2841 (reference gene) were measured by RT-qPCR using the TaqMan chemistry in the strains listed below at 12 and 15 hours of growth at 25°C (starting from synchronized L1 population). Condition Strain code Genotype wild type N2 wild type let-7(∆BS) HW3343 let-7(xe336[GGAGmut]) X lin-28(0) HW2705 lin-28(xe192) I Fig. S6A: western blot Uncropped images of the western blot get used in Figure S6A. The western blot is showing the levels of both LIN28 isoforms, with size differences due to varying tag sizes in different genetic backgrounds. Note that lin-28(∆a) is not further analyzed in this study. As an antibody specificity control, lin-28(0) animals, which lack LIN28 protein, were included. Animals were grown at 25°C and collected at 3 and 6 h of development after plating of synchronized L1s. Protein lysates were probed using anti-LIN28 and anti-actin antibodies. *: LIN28 is C-terminally FLAG::HA-tagged. This entry includes a PDF file containing compiled, uncropped, and annotated gel images (S1_raw_images.pdf), as well as four TIFF files with unprocessed gel images (FigS6A_act-1_5s_marker_channel.tif, FigS6A_act-1_5s_POI_channel.tif, FigS6A_LIN-28_1s_marker_channel.tif, FigS6A_LIN-28_1s_POI_channel.tif). Condition Strain code Genotype wild type N2 wild type wild type* HW1792 lin-28(xe59[lin-28::flag::ha]) I lin-28(Δb)* HW3381 lin-28(xe344xe59[lin-28::flag::ha with del_exon1B]) I lin-28::large_tag HW2805 lin-28(xe197[lin-28::linker::aid::3xflag::tev::gfp::linker::hibit]) I lin-28(0) HW2705 lin-28(xe192) I Fig. S6B: qPCR let-7 lin-46 timecourse Raw qPCR data supporting the Fig. S6B. Expression levels of mature let-7 miRNA (left) and lin-46 mRNA (right) measured by RT-qPCR in animals grown at 25°C and collected hourly from 12 to 17 hours of development. Expression levels at each time point were first normalized to the levels of a reference gene (small nucleolar RNA sn2841 for let-7, actin mRNA for lin-46) and then to wild type at 12 h. Condition Strain code Genotype wild type N2 wild type wild type* HW1792 lin-28(xe59[lin-28::flag::ha]) I lin-28(0) HW2705 lin-28(xe192) I lin-28(∆b) HW3381 lin-28(xe344xe59[lin-28::flag::ha with exon1B(Δ)]) I lin-28::large_tag HW2805 lin-28(xe197[lin-28::linker::aid::3xflag::tev::gfp::linker::hibit]) I Fig. S7A: Pvl phenotype quantification II Quantification of the protruding vulva (Pvl) phenotype of adult animals of indicated genotypes. Animals were grown at 20°C and examined at 54 h of development. N = 4 technical replicates. Condition Strain code Genotype wild type N2 wild type lin-28(0) HW2705 lin-28(xe192) I lin-28(n719) HW11 lin-28(n719) I let-7(++) HW1909 xeSi287[let-7p::let-7] V lin-46(++) HW3531 lin-46(xe370) V let-7(++) lin-46(++) HW3808 xeSi287[let-7p::let-7] lin-46(xe370[GGAG_mut]) V lin-28::large_tag HW2805 lin-28(xe197) I lin-28::large_tag; let-7(++) HW3758 lin-28(xe197) I; xeSi287[let-7p::let-7] V lin-28::large_tag; lin-46(++) HW3685 lin-28(xe197) I; lin-46(xe370) V lin-28::large_tag; let-7(++) lin-46(++) HW3883 lin-28(xe197) I; xeSi287[let-7p::let-7] lin-46(xe370) V