Reporting Checklist for Nature Neuroscience

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1 Corresponding Author: Manuscript Number: Manuscript Type: xiangdong fu, yuanchao xue NNA52804A Article Reporting Checklist Nature Neuroscience # Main Figures: 5 # Supplementary Figures: 7 # Supplementary Tables: # Supplementary Videos: none This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. For more inmation, please read Reporting Life Sciences Research. Please note that in the event of publication, it is mandatory that authors include all relevant methodological and statistical inmation in the manuscript. Statistics reporting, by example example Please specify the following inmation each panel reporting quantitative data, and where each item is reported (section, e.g. Results, & paragraph number). Each should ideally contain an exact sample size (n) each experimental group/condition, where n is an exact number and not a range, a clear definition of how n is defined ( example x cells from x slices from x animals from x litters, collected over x days), a description of the statistical used, the results of the s, any descriptive statistics and clearly defined error bars if applicable. For any using custom statistics, please indicate the used and stats obtained each experiment. Each should include a statement of how many times the experiment shown was replicated in the lab; the details of sample collection should be sufficiently clear so that the replicability of the experiment is obvious to the reader. For reported in the text but not in the s, please use the paragraph number instead of the number. Note: Mean and standard deviation are not appropriate on small samples, and plotting independent data points is usually more inmative. When technical replicates are reported, error and significance measures reflect the experimental variability and not the variability of the biological process; it is misleading not to state this clearly. FIGURE NUMBER 1a results, TEST USED WHICH TEST? oneway ANOVA t Results EXACT VALUE 9, 9, 10, 15 n DEFINED? mice from at least litters/group 15 slices from 10 mice Methods para 8 Results DESCRIPTIVE STATS (AVERAGE, VARIANCE) REPORTED? error bars are mean / SEM error bars are mean / SEM Results P VALUE EXACT VALUE p = p = Results DEGREES OF FREEDOM & F/t/z/R/ETC VALUE VALUE F(, 6) = 2.97 t(28) = Results 1

2 FIGURE NUMBER 1a 1b 1c 1e 2f2k a b d k 4e TEST USED WHICH TEST? twotailed For only, no needed only, no needed only, no needed No needed only only No needed twotailed EXACT VALUE 5,5 6, 24 f(10/10), g(8/11), h(10/10), ik(9/11), 2 d(11/11), e(10/11), f(5/12), g(8/10), ik(11/12) 2, n DEFINED? n=5 20x random fields shctrl and shptb n=6 random 20x fields on 86 year female n= random 20x fields on 42 year male 2 to 4 randomly chosen fields were used quantification at different conditions all data points are based on independent biological repeats number of cells showing recorded activity versus total number of cells examined data from 2 biological repeats n= independent both shctrl and shptb/nptb number of cells showing recorded activity versus total number of cells examined n= independent in all boxes, except box 10 in the lower panel where n=2 due to a failed sample DESCRIPTIVE STATS (AVERAGE, VARIANCE) REPORTED? Para. 6 Para. 6 Para. 7 Para. 8 Para. 12 Para. 1 2f2k, and Para. 1 Para. 15 Para. 18 dk, and P VALUE EXACT VALUE P= For mir124 in upper panel: box 2/8: P= 0.977, box 10/4: P= , box 12/6: P=0.49; For mir9 in lower panel: box 2/8: P=0.0795, box 10/4: P=0.002, box 6/12: P= DEGREES OF FREEDOM & F/t/z/R/ETC VALUE VALUE df=8 t=6.87 For mir124 in upper panel: box 2/8: df=4, t= 0.004, box 10/4: df=4, t=2.65, box 12/6: df=4, t=1.059, For mir9 in lower panel: box 2/8: df=4, t= 2.9, box 4/10: df=, t=8.58, box 6/12: df=4, t=4.577 Para. 6 Para. 6 Para. 7 Para. 8 Para. 12 Para. 1 Para. 1 Para. 15 Para. 18 2

3 4f 4g 4h 5b twotailed twotailed twotailed Twotailed,5,6 2, 2, 2, n= independent all boxes, except box 7 in lower panel where n=5, and box 8 and 9 in lower panel where n=6 n= independent all boxes except box 2 and 7 where n=2 n= independent except box 2 in the right panel where n=2 n=2 independent nonspecific control (NC), and n= independent the remaining two conditions Para. 18 Para. 19 Para. 19 For upper panel: P=0.275, box 1/: P=0.182, box 4/5: P=0.024, box 4/6: P=0.077, box 7/8: P=0.146, box 7/9: P=0.117 For lower panel: P=0.0152, box 1/: P=0.110, box 4/5: P= , box 4/6: P= , box 7/8: P=0.014, box 7/9: P= P= , box /4: P= , box5/6: P= , box 7/8: P= , box 9/10: P= , box 11/12: P=0.0047, box 2/4: P=0.0122, box 4/6: P= For mir9 in left panel: P=0.0249, box 2/: P= , box 1/: p= For mir124 in right panel: P= , box 2/: P= , box 1/: P= P=0.064, box 1/: P= For upper panel: df=4, t=1.26, box 1/: df=4, t=1.615, box 4/5: df=4, t=.529, box 4/6: df=4, t=.059, box 7/8: df=4 t=1.802, box 7/9: df=4, t=1.995 For lower panel: df=4, t=4.069, box 1/: df=4, t=2.051, box 4/5: df=4, t=5.21, box 4/6: df=4, t=9.56, box 7/8: df=9 t=.066, box 7/9: df=9, t=8.07 df=, t=6.484, box /4: df=4, t=16.616, box 5/6: df=4, t=12.795, box 7/,8: df= t=1.218, box 9/10: df=4, t=0.228, box 11/12: df=4, t=5.684, box 2/4: df=, t=5.446, box 4/6: df=4, t=5.154,for mir9 in left panel: df=4, t=.496, box 2/: df=4, t=8.85, box 1/: df=4, t= For mir124 in right panel: df=, t=11.886, box 2/: df=, t=9.15, box 1/: df=4, t= df=, t=.614 box 1/: df=, t= 5.28 Para. 18 Para. 19 Para. 19

4 5c 5d 5e 5f 5g s2c sa s5a twotailed only only twotailed twotailed twotailed twotailed 2, 2, n= independent the first two conditions and n=2 independent the third condition n= independent n= independent n= independent n= independent all boxes except box 7 where n=2. n= independent n= independent n= independent Para. 21 Para. 11 Para. 1 P=0.0060, box 1/: P= P=0.005, box 1/: P= P=0.017, box 2/: P= , box 2/4: P=0.0006, box 5/6: P=0.0042, box 7/8: P= , box 9/10: P= Nonspecific (NS): P=0.564, 1#: P= , 2#: P=0.0042, #: P= , 4#: P= Ascl1: P= , Myt1l: P= , NeuroD1: P= , Olig2: P= , Brn2: P=0.020, mir124: P= P= df=4, t=6.124, box 1/: df= t=6.861 df=4, t=5.50, box 1/: df=4, t=4.910 df=4, t=4.197, box 2/: df=4, t= box 2/4: df=4, t=6.409, box 5/6: df=4, t=5.827, box 7/8: df=, t=9.095, box 9/10: df=4, t=4.057 NS: df=4, t=0.628, 1#: df=4, t=16.752, 2#: df=4, t=5.872, #: df=4, t=27.11, 4#: df=4, t=9.992 Ascl1: df=4, t=5.6, Myt1l : df=4, t=11.605, NeuroD1: df=4, t=4.602, Olig2: df=4, t=12.2,, Brn2: df=4, t=.77 mir124: df=4 t=5.812 df=4, t=1.208 Para. 21 Para. 11 Para. 1 4

5 Representative s 1. Are any representative images shown (including Western blots and immunohistochemistry/staining) in the paper? If so, what (s)? 2. For each representative image, is there a clear statement of how many times this experiment was successfully repeated and a discussion of any limitations in repeatability? If so, where is this reported (section, paragraph #)? Statistics and general methods 1. Is there a justification of the sample size? If so, how was it justified? Even if no sample size calculation was permed, authors should report why the sample size is adequate to measure their effect size. 2. Are statistical s justified as appropriate every? a. If there is a section summarizing the statistical methods in the methods, is the statistical each experiment clearly defined? b. Do the data meet the assumptions of the specific statistical you chose (e.g. normality a parametric )? Where is this described (section, paragraph #)? c. Is there any estimate of variance within each group of data? Is the variance similar between groups that are being statistically compared? Where is this described (section, paragraph #)? Yes. Western blots: Figure 2b,2c; Figure s2d; Figure s4e and s4f; Figure5b, 5c, 5d, 5e, 5f and 5h; Figure S5a and S5b. Immunostaining/immunofluresence: Figure 1a, 1b, 1c and 1d; Figure 2a, 2e and 2i; Figure c and h; Figure 4a and 4b; Figure S1a, S1c, S1d, and S1f; Figure S2a; Figure Sb; Figure S4a, S4b, S4c, and S4i. Phase contrast images: Figure S1e. Ca imaging: Figure Sc. Yes. We stated the number of random fields selected quantitative analysis as described in the of each. Sample sizes were described in the of each. The sample size (in most cases, at least ) allows us to achieve at least 80% power (standard power) to detect the difference with 95% confidence. However, as we specifically indicated in the checklist, some samples were lost during the experiment, but the remaining 2 samples still allowed meaningful to achieve statistical significance Yes. We described the justification in the Methods (Statistical Analysis, Page 8/9, Paragraph 2). To each, the statistical analysis was described in the corresponding and justified to detect the difference with 95% confidence. Yes. We summarized in Methods at Page 8/9, para2. Each statistical was described in the corresponding. For statistical s reported in each, the data meet the assumption of normal distribution and are appropriate ts. Yes. For statistical es reported in Figs.15, there is an estimate of variation within each group of data. The variance is similar between the groups statistically compared, which described in Methods at Page 8/9, paragraph 2. 5

6 d. Are s specified as one or twosided? Yes. twotailed student t were used and specified in in Methods at Page 8/9, paragraph 2. e. Are there adjustments multiple s? No. Are criteria excluding data points reported? Was this criterion established prior to data collection? Where is this described (section, paragraph #)? 4. Define the method of randomization used to assign subjects (or samples) to the experimental groups and to collect and process data. If no randomization was used, state so. Where does this appear (section, paragraph #)? 5. Is a statement of the extent to which investigator knew the group allocation during the experiment and in assessing outcome included? If no blinding was done, state so. 6. For in live vertebrates, is a statement of compliance with ethical guidelines/regulations included? 7. Is the species of the animals used reported? 8. Is the strain of the animals (including background strains of KO/ transgenic animals used) reported? 9. Is the sex of the animals/subjects used reported? 10. Is the age of the animals/subjects reported? 11. For animals housed in a vivarium, is the light/dark cycle reported? 12. For animals housed in a vivarium, is the housing group (i.e. number of animals per cage) reported? No data points were excluded from the analysis. All data collection and analyses were permed by experimenters blind to the treatment conditions. We make the statement in method part at Page 8/9, para2.. Yes. on page 1, last paragraph. Yes. on page 1, last paragraph. Yes. on page 1, last paragraph. 6

7 1. For behavioral, is the time of day reported (e.g. light or dark cycle)? 14. Is the previous history of the animals/subjects (e.g. prior drug administration, surgery, behavioral ing) reported? a. If multiple behavioral s were conducted in the same group of animals, is this reported? 15. If any animals/subjects were excluded from analysis, is this reported? Reagents a. How were the criteria exclusion defined? Where is this described (section, paragraph #)? b. Specify reasons any discrepancy between the number of animals at the beginning and end of the study. Where is this described (section, paragraph #)? 1. Have antibodies been validated use in the system under study (assay and species)? a. Is antibody catalog number given? Where does this appear (section, paragraph #)? b. Where were the validation data reported (citation, supplementary inmation, Antibodypedia)? Where does this appear (section, paragraph #)? 2. If cell lines were used to reflect the properties of a particular tissue or disease state, is their source identified? a. Were they recently authenticated? Where is this inmation reported (section, paragraph #)? Yes Yes, see Methods at page 5 Brn2 antibody was validated at Figure S4f and page 12 at para 2. For other antibodies, validation was reported from the companies where we purchased the antibodies or from cited literatures (page 5). Yes, see Methods at page 0 and Results at page 5, para 2. Two human adult fibroblast lines were used in this study, one is from ATCC and the other is from Dr. John Ravits, which has not been reported yet. 7

8 Data deposition Data deposition in a public repository is mandatory : a. Protein, DNA and RNA sequences b. Macromolecular structures c. Crystallographic data small molecules d. Microarray data Deposition is strongly recommended many other datasets which structured public repositories exist; more details on our data policy are available here. We encourage the provision of other source data in supplementary inmation or in unstructured repositories such as Figshare and Dryad. We encourage publication of Data Descriptors (see Scientific Data) to maximize data reuse. 1. Are accession codes deposit dates provided? Computer code/software Yes. RNAseq and Brn2 ChIPseq data has been deposited under the exccession number of GSE Any custom algorithm/software that is central to the methods must be supplied by the authors in a usable and readable m readers at the time of publication. However, referees may ask this inmation at any time during the review process. 1. Identify all custom software or scripts that were required to conduct the study and where in the procedures each was used. 2. If computer code was used to generate results that are central to the paper's conclusions, include a statement in the Methods section under "Code availability" to indicate whether and how the code can be accessed. Include version inmation as necessary and any restrictions on availability. Human subjects 1. Which IRB approved the protocol? Where is this stated (section, paragraph #)? 2. Is demographic inmation on all subjects provided?. Is the number of human subjects, their age and sex clearly defined? 4. Are the inclusion and exclusion criteria (if any) clearly specified? 8

9 5. How well were the groups matched? Where is this inmation described (section, paragraph #)? 6. Is a statement included confirming that inmed consent was obtained from all subjects? 7. For publication of patient photos, is a statement included confirming that consent to publish was obtained? fmri studies For papers reporting functional imaging (fmri) results please ensure that these minimal reporting guidelines are met and that all this inmation is clearly provided in the methods: 1. Were any subjects scanned but then rejected the analysis after the data was collected? a. If yes, is the number rejected and reasons rejection described? 2. Is the number of blocks, trials or experimental units per session and/ or subjects specified?. Is the length of each trial and interval between trials specified? 4. Is a blocked, eventrelated, or mixed design being used? If applicable, please specify the block length or how the eventrelated or mixed design was optimized. 5. Is the task design clearly described? 6. How was behavioral permance measured? 7. Is an ANOVA or factorial design being used? 8. For data acquisition, is a whole brain scan used? If not, state area of acquisition. a. How was this region determined? 9

10 9. Is the field strength (in Tesla) of the MRI system stated? a. Is the pulse sequence type (gradient/spin echo, EPI/spiral) stated? b. Are the fieldofview, matrix size, slice thickness, and TE/TR/ flip angle clearly stated? 10. Are the software and specific parameters (model/functions, smoothing kernel size if applicable, etc.) used data processing and preprocessing clearly stated? 11. Is the coordinate space the anatomical/functional imaging data clearly defined as subject/native space or standardized stereotaxic space, e.g., original Talairach, MNI05, ICBM152, etc? Where (section, paragraph #)? 12. If there was data normalization/standardization to a specific space template, are the type of transmation (linear vs. nonlinear) used and image types being transmed clearly described? Where (section, paragraph #)? 1. How were anatomical locations determined, e.g., via an automated labeling algorithm (AAL), standardized coordinate database (Talairach daemon), probabilistic atlases, etc.? 14. Were any additional regressors (behavioral covariates, motion etc) used? 15. Is the contrast construction clearly defined? 16. Is a mixed/random effects or fixed inference used? a. If fixed effects inference used, is this justified? 17. Were repeated measures used (multiple measurements per subject)? a. If so, are the method to account within subject correlation and the assumptions made about variance clearly stated? 18. If the threshold used inference and visualization in s varies, is this clearly stated? 19. Are statistical inferences corrected multiple s? a. If not, is this labeled as uncorrected? 10

11 20. Are the results based on an ROI (region of interest) analysis? a. If so, is the rationale clearly described? b. How were the ROI s defined (functional vs anatomical localization)? 21. Is there correction multiple s within each voxel? 22. For clusterwise significance, is the clusterdefining threshold and the corrected significance level defined? Additional comments Additional Comments 11