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Figure S1 the Pipeline of Calling and Filtering Rare Variants from RNA-Seq Data in This Study

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BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Ann Rheum Dis Figure S1 The pipeline of calling and filtering rare variants from RNA-seq data in this study. 1 Meng X, et al. Ann Rheum Dis 2021; 80:626–631. doi: 10.1136/annrheumdis-2020-218359 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Ann Rheum Dis Figure S2 Validation of variants called from RNA-seq by Sanger sequencing in an independent cohort of three samples. Sanger sequencing chromatograms of the validated variants among 10 variants randomly selected from each samples. Sample1 Sample2 2 Meng X, et al. Ann Rheum Dis 2021; 80:626–631. doi: 10.1136/annrheumdis-2020-218359 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Ann Rheum Dis Sample3 3 Meng X, et al. Ann Rheum Dis 2021; 80:626–631. doi: 10.1136/annrheumdis-2020-218359 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Ann Rheum Dis Figure S3 The sample quality control filtering based on the number of rare variants carried by each sample. The number of rare variants called from RNA-seq of each sample was plotted and those outside of the linear phase of the distribution were filtered out. The number of variants in the linear phase is shown in orange and the number of variants which is either too low or too high in the exponential phase is shown in grey, indicating the excluded samples. 250 200 150 100 variant number variant 50 0 0 100 200 300 400 500 600 sample ID 4 Meng X, et al. Ann Rheum Dis 2021; 80:626–631. doi: 10.1136/annrheumdis-2020-218359 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Ann Rheum Dis Figure S4 The distribution of mutation types among the 63 recurrent rare variants found in our study. SNVs 2% 5% missense_NA 30% missense_D missense_T 63% stoploss/stopgain_NA missense_NA: rare missense SNVs with SIFT functional prediction not available; missense_D: rare missense SNVs with SIFT functional prediction to be deleterious; missense_T: rare missense SNVs with SIFT functional prediction to be tolerant; stoploss/stopgain_NA: SNVs with a gene truncating effect (stop gain or stop loss) with SIFT functional prediction not available. 5 Meng X, et al. Ann Rheum Dis 2021; 80:626–631. doi: 10.1136/annrheumdis-2020-218359 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Ann Rheum Dis Figure S5. The expression level of genes with recurrent deleterious rare variants based on data in DICE. (https://dice-database.org/) A. polyarticular JIA 6 Meng X, et al. Ann Rheum Dis 2021; 80:626–631. doi: 10.1136/annrheumdis-2020-218359 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Ann Rheum Dis B. oligoarticular JIA C. systemic JIA DICE:Database of Immune Cell Expression; TPM:Transcripts per million. 7 Meng X, et al. Ann Rheum Dis 2021; 80:626–631. doi: 10.1136/annrheumdis-2020-218359 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Ann Rheum Dis Figure S6 The expression level of genes with recurrent deleterious rare variants based on in HPA (https://www.proteinatlas.org/) A. polyarticular JIA 8 Meng X, et al. Ann Rheum Dis 2021; 80:626–631. doi: 10.1136/annrheumdis-2020-218359 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Ann Rheum Dis B. oligoarticular JIA C. systemic JIA HPA: Human protein Atlas; pTPM: Protein- transcripts per million. 9 Meng X, et al. Ann Rheum Dis 2021; 80:626–631. doi: 10.1136/annrheumdis-2020-218359 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Ann Rheum Dis Figure S7 The differentially expressed genes in the meta-analysis of polyarticular and oligoarticular subtypes of three RNA-seq datasets (|FC|>2, adjusted P-value <0.05). Volcano plot showing the gene differential expression analysis of three RNA-seq datasets. The vertical lines correspond to 2.0-fold up and down, respectively, and the horizontal line represents adjust-p value of 0.05. The red points in the plot represent the 43 significantly up-regulated genes; and the blue points represent the 4 significantly down-regulated genes. 10 Meng X, et al. Ann Rheum Dis 2021; 80:626–631. doi: 10.1136/annrheumdis-2020-218359 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Ann Rheum Dis Figure S8 Volcano plots showing the results of pathway over-representation analysis. It shows the log of the FDR versus the enrichment ratio for all the pathways in the KEGG database, highlighting the degree by which the significant pathways stand out from the background. The size and color of the dot is proportional to the number of input genes falling into each pathway. (A) Pathway analysis of 63 genes with recurrent rare variants; (B) Pathway analysis of 138 differentially expressed genes of polyarticular and oligoarticular subtypes of JIA. A B 11 Meng X, et al. Ann Rheum Dis 2021; 80:626–631. doi: 10.1136/annrheumdis-2020-218359 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Ann Rheum Dis Table S1 The fraction of rare variants called from RNA-sequencing data were detected in whole exome sequencing (WES) data of each of the 100 randomly selected GTEx samples. RNA-seq WES Fraction RNA-seq WES Fraction SRR1069024 SRR2167400 72.70% SRR1361368 SRR2167124 60.80% SRR1071931 SRR2165980 66.20% SRR1363611 SRR2165743 66.70% SRR1072345 SRR2168332 77.40% SRR1366543 SRR2165999 80.40% SRR1072529 SRR2166291 88.00% SRR1368529 SRR2167121 75.80% SRR1072871 SRR2167072 83.10% SRR1370742 SRR2165890 83.30% SRR1074646 SRR2167359 62.70% SRR1378026 SRR2167957 82.10% SRR1076511 SRR2165120 69.20% SRR1381435 SRR2168744 82.20% SRR1077503 SRR2166156 80.90% SRR1381481 SRR2166915 87.80% SRR1079259 SRR2165448 60.40% SRR1382615 SRR2168325 72.20% SRR1079660 SRR2167414 79.70% SRR1383855 SRR2156637 78.30% SRR1080998 SRR2167470 80.40% SRR1386967 SRR2166500 76.50% SRR1083432 SRR1343762 76.70% SRR1388081 SRR2170921 64.90% SRR1083657 SRR2167152 62.20% SRR1389133 SRR1310297 74.00% SRR1084577 SRR2165457 90.20% SRR1392547 SRR1348058 76.60% SRR1084720 SRR2164914 77.10% SRR1396191 SRR2167760 85.50% SRR1087654 SRR2167068 68.80% SRR1397673 SRR2165117 85.70% SRR1090508 SRR2166372 91.30% SRR1400032 SRR2163570 82.20% SRR1091452 SRR2164811 62.70% SRR1400054 SRR2163573 82.50% SRR1092422 SRR2166301 100% SRR1407758 SRR2157513 78.80% SRR1096571 SRR1305587 75.80% SRR1409510 SRR2167950 84.20% SRR1099260 SRR2167942 90.70% SRR1413892 SRR2166259 86.50% SRR1099648 SRR2167756 91.40% SRR1416560 SRR2167440 85.50% SRR1099744 SRR2165963 72.70% SRR1420669 SRR2170534 69.70% SRR1101324 SRR2166998 77.90% SRR1433044 SRR2167335 78.70% SRR1101787 SRR2167293 70.50% SRR1433860 SRR2166939 86.80% SRR1102031 SRR2166948 70.20% SRR1435833 SRR2164488 68.90% SRR1310412 SRR2165954 87.10% SRR1440224 SRR2165753 77.80% SRR1311419 SRR2166173 88.50% SRR1440603 SRR2167421 91.70% SRR1311644 SRR2167963 85.30% SRR1444852 SRR2165960 78.60% SRR1312598 SRR2170783 60.70% SRR1446244 SRR2170487 65.90% SRR1314916 SRR2167408 66.70% SRR1446850 SRR2167131 75.00% SRR1320137 SRR2166827 71.20% SRR1447523 SRR2167496 90.40% SRR1322619 SRR2165570 64.10% SRR1457574 SRR2167277 84.60% SRR1326536 SRR2167467 85.50% SRR1459739 SRR2166269 70.30% SRR1328407 SRR2164694 94.40% SRR1464618 SRR2165024 69.10% SRR1329047 SRR2167309 89.60% SRR1468850 SRR2163078 76.90% SRR1331311 SRR2165452 67.90% SRR1474839 SRR2165523 84.60% SRR1331684 SRR2170126 86.00% SRR1475930 SRR2166052 72.60% SRR1332007 SRR2164492 84.80% SRR1476207 SRR1306141 66.00% SRR1333570 SRR2165222 93.50% SRR1476300 SRR2166515 81.50% SRR1337540 SRR2166369 78.30% SRR1477991 SRR2167387 69.00% 12 Meng X, et al. Ann Rheum Dis 2021; 80:626–631. doi: 10.1136/annrheumdis-2020-218359 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Ann Rheum Dis SRR1338880 SRR2167707 62.50% SRR1480789 SRR2167332 90.50% SRR1339619 SRR1310396 60.00% SRR1481060 SRR2164704 88.00% SRR1345266 SRR2156686 66.00% SRR1481351 SRR1308426 78.80% SRR1345541 SRR2155763 81.20% SRR1486021 SRR2164933 62.70% SRR1345847 SRR2170211 63.20% SRR1488261 SRR1307843 84.10% SRR1349032 SRR2167437 62.70% SRR1489116 SRR2157311 78.90% SRR1349276 SRR2166865 60.90% SRR1489926 SRR2164748 83.70% SRR1352530 SRR1307722 79.40% SRR1491332 SRR2165363 88.60% SRR1355102 SRR2156394 73.00% SRR1498313 SRR2170789 88.90% RNA-seq= the SRR ID of the RNA-seq file; WES= the SRR ID of the WES file for the same sample as the RNA-seq file; fraction= the fraction of rare variants called from RNA-seq data was detected by the WES data 13 Meng X, et al.
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    https://www.alphaknockout.com Mouse Epb42 Knockout Project (CRISPR/Cas9) Objective: To create a Epb42 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Epb42 gene (NCBI Reference Sequence: NM_013513 ; Ensembl: ENSMUSG00000023216 ) is located on Mouse chromosome 2. 13 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 13 (Transcript: ENSMUST00000102490). Exon 2~5 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Homozygotes for a targeted null mutation exhibit erythrocytic abnormalities including mild spherocytosis, altered ion transport, and dehydration. Exon 2 starts from about 0.53% of the coding region. Exon 2~5 covers 31.07% of the coding region. The size of effective KO region: ~5547 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 5 13 Legends Exon of mouse Epb42 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 1985 bp section upstream of Exon 2 is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 1230 bp section downstream of Exon 5 is aligned with itself to determine if there are tandem repeats.
  • Supplementary Material

    Supplementary Material

    BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Neurol Neurosurg Psychiatry Page 1 / 45 SUPPLEMENTARY MATERIAL Appendix A1: Neuropsychological protocol. Appendix A2: Description of the four cases at the transitional stage. Table A1: Clinical status and center proportion in each batch. Table A2: Complete output from EdgeR. Table A3: List of the putative target genes. Table A4: Complete output from DIANA-miRPath v.3. Table A5: Comparison of studies investigating miRNAs from brain samples. Figure A1: Stratified nested cross-validation. Figure A2: Expression heatmap of miRNA signature. Figure A3: Bootstrapped ROC AUC scores. Figure A4: ROC AUC scores with 100 different fold splits. Figure A5: Presymptomatic subjects probability scores. Figure A6: Heatmap of the level of enrichment in KEGG pathways. Kmetzsch V, et al. J Neurol Neurosurg Psychiatry 2021; 92:485–493. doi: 10.1136/jnnp-2020-324647 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Neurol Neurosurg Psychiatry Appendix A1. Neuropsychological protocol The PREV-DEMALS cognitive evaluation included standardized neuropsychological tests to investigate all cognitive domains, and in particular frontal lobe functions. The scores were provided previously (Bertrand et al., 2018). Briefly, global cognitive efficiency was evaluated by means of Mini-Mental State Examination (MMSE) and Mattis Dementia Rating Scale (MDRS). Frontal executive functions were assessed with Frontal Assessment Battery (FAB), forward and backward digit spans, Trail Making Test part A and B (TMT-A and TMT-B), Wisconsin Card Sorting Test (WCST), and Symbol-Digit Modalities test.
  • Nuclear PTEN Safeguards Pre-Mrna Splicing to Link Golgi Apparatus for Its Tumor Suppressive Role

    Nuclear PTEN Safeguards Pre-Mrna Splicing to Link Golgi Apparatus for Its Tumor Suppressive Role

    ARTICLE DOI: 10.1038/s41467-018-04760-1 OPEN Nuclear PTEN safeguards pre-mRNA splicing to link Golgi apparatus for its tumor suppressive role Shao-Ming Shen1, Yan Ji2, Cheng Zhang1, Shuang-Shu Dong2, Shuo Yang1, Zhong Xiong1, Meng-Kai Ge1, Yun Yu1, Li Xia1, Meng Guo1, Jin-Ke Cheng3, Jun-Ling Liu1,3, Jian-Xiu Yu1,3 & Guo-Qiang Chen1 Dysregulation of pre-mRNA alternative splicing (AS) is closely associated with cancers. However, the relationships between the AS and classic oncogenes/tumor suppressors are 1234567890():,; largely unknown. Here we show that the deletion of tumor suppressor PTEN alters pre-mRNA splicing in a phosphatase-independent manner, and identify 262 PTEN-regulated AS events in 293T cells by RNA sequencing, which are associated with significant worse outcome of cancer patients. Based on these findings, we report that nuclear PTEN interacts with the splicing machinery, spliceosome, to regulate its assembly and pre-mRNA splicing. We also identify a new exon 2b in GOLGA2 transcript and the exon exclusion contributes to PTEN knockdown-induced tumorigenesis by promoting dramatic Golgi extension and secretion, and PTEN depletion significantly sensitizes cancer cells to secretion inhibitors brefeldin A and golgicide A. Our results suggest that Golgi secretion inhibitors alone or in combination with PI3K/Akt kinase inhibitors may be therapeutically useful for PTEN-deficient cancers. 1 Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025, China. 2 Institute of Health Sciences, Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences and SJTU-SM, Shanghai 200025, China.