{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T10:31:24Z","timestamp":1791973884461,"version":"4.4.0"},"reference-count":68,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2024,6,13]],"date-time":"2024-06-13T00:00:00Z","timestamp":1718236800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"Major Program of National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["81827901"],"award-info":[{"award-number":["81827901"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"Major Program of National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["91749209"],"award-info":[{"award-number":["91749209"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["81827901"],"award-info":[{"award-number":["81827901"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["91749209"],"award-info":[{"award-number":["91749209"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Paraformaldehyde (PFA) fixation is the preferred method for preserving tissue architecture for anatomical and pathological observations. Meanwhile, PFA reacts with the amine groups of biomolecules to form chemical cross-linking, which preserves RNA within the tissue. This has great prospects for RNA sequencing to characterize the molecular underpinnings after anatomical and pathological observations. However, RNA is inaccessible due to cross-linked adducts forming between RNA and other biomolecules in prolonged PFA-fixed tissue. It is also difficult to perform reverse transcription and PCR, resulting in low sequencing sensitivity and reduced reproducibility. Here, we developed a method to perform RNA sequencing in PFA-fixed tissue, which is easy to use, cost-effective, and allows efficient sample multiplexing. We employ cross-link reversal to recover RNA and library construction using random primers without artificial fragmentation. The yield and quality of recovered RNA significantly increased through our method, and sequencing quality metrics and detected genes did not show any major differences compared with matched fresh samples. Moreover, we applied our method for gene expression analysis in different regions of the mouse brain and identified unique gene expression profiles with varied functional implications. We also find significant dysregulation of genes involved in Alzheimer\u2019s disease (AD) pathogenesis within the medial septum (MS)\/vertical diagonal band of Broca (VDB) of the 5\u00d7FAD mouse brain. Our method can thus increase the performance of high-throughput RNA sequencing with PFA-fixed samples and allows longitudinal studies of small tissue regions isolated by their in situ context.<\/jats:p>","DOI":"10.3390\/ijms25126504","type":"journal-article","created":{"date-parts":[[2024,6,14]],"date-time":"2024-06-14T03:52:53Z","timestamp":1718337173000},"page":"6504","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Streamlined Full-Length Total RNA Sequencing of Paraformaldehyde-Fixed Brain Tissues"],"prefix":"10.3390","volume":"25","author":[{"given":"Bingqing","family":"Ji","sequence":"first","affiliation":[{"name":"Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China"},{"name":"MoE Key Laboratory for Biomedical Photonics, Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiale","family":"Chen","sequence":"additional","affiliation":[{"name":"Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China"},{"name":"MoE Key Laboratory for Biomedical Photonics, Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hui","family":"Gong","sequence":"additional","affiliation":[{"name":"Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China"},{"name":"MoE Key Laboratory for Biomedical Photonics, Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China"},{"name":"Research Unit of Multimodal Cross Scale Neural Signal Detection and Imaging, HUST-Suzhou Institute for Brainsmatics, JITRI, Chinese Academy of Medical Sciences, Suzhou 215125, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiangning","family":"Li","sequence":"additional","affiliation":[{"name":"Research Unit of Multimodal Cross Scale Neural Signal Detection and Imaging, HUST-Suzhou Institute for Brainsmatics, JITRI, Chinese Academy of Medical Sciences, Suzhou 215125, China"},{"name":"Key Laboratory of Biomedical Engineering of Hainan Province, School of Biomedical Engineering, Hainan University, Haikou 570228, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,6,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3388","DOI":"10.1021\/ja908282f","article-title":"Structural Characterization of Formaldehyde-Induced Cross-Links Between Amino Acids and Deoxynucleosides and Their Oligomers","volume":"132","author":"Lu","year":"2010","journal-title":"J. Am. Chem. Soc."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Qidwai, K., Afkhami, M., and Day, C.E. (2014). The Pathologist\u2019s Guide to Fixatives, Springer.","DOI":"10.1007\/978-1-4939-1050-2_2"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"713","DOI":"10.1146\/annurev-cellbio-111315-125001","article-title":"Chemical Principles in Tissue Clearing and Staining Protocols for Whole-Body Cell Profiling","volume":"32","author":"Tainaka","year":"2016","journal-title":"Annu. Rev. Cell Dev. Biol."},{"key":"ref_4","unstructured":"Suvarna, K.S., Layton, C., and Bancroft, J.D. (2018). Bancroft\u2019s Theory and Practice of Histological Techniques, Elsevier."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"400","DOI":"10.4103\/0973-029X.102496","article-title":"Chemical and physical basics of routine formaldehyde fixation","volume":"16","author":"Thavarajah","year":"2012","journal-title":"J. Oral Maxillofac. Pathol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"4115","DOI":"10.1016\/j.cell.2021.07.009","article-title":"Basic principles of hydrogel-based tissue transformation technologies and their applications","volume":"184","author":"Choi","year":"2021","journal-title":"Cell"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1682","DOI":"10.1038\/nprot.2014.123","article-title":"Advanced CLARITY for rapid and high-resolution imaging of intact tissues","volume":"9","author":"Tomer","year":"2014","journal-title":"Nat. Protoc."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"332","DOI":"10.1038\/nature12107","article-title":"Structural and molecular interrogation of intact biological systems","volume":"497","author":"Chung","year":"2013","journal-title":"Nature"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"4436","DOI":"10.1093\/nar\/27.22.4436","article-title":"Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples","volume":"27","author":"Masuda","year":"1999","journal-title":"Nucleic Acids Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"282","DOI":"10.1016\/j.jmoldx.2011.01.010","article-title":"The effect of formaldehyde fixation on RNA: Optimization of formaldehyde adduct removal","volume":"13","author":"Evers","year":"2011","journal-title":"J. Mol. Diagn."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"21418","DOI":"10.1038\/srep21418","article-title":"Straightforward and sensitive RT-qPCR based gene expression analysis of FFPE samples","volume":"6","author":"Zeka","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Channathodiyil, P., and Houseley, J. (2021). Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry. PLoS ONE, 16.","DOI":"10.1371\/journal.pone.0240769"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2501","DOI":"10.1038\/s41596-018-0045-2","article-title":"Barcoded solid-phase RNA capture for Spatial Transcriptomics profiling in mammalian tissue sections","volume":"13","author":"Mollbrink","year":"2018","journal-title":"Nat. Protoc."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1002\/(SICI)1098-2825(1997)11:1<2::AID-JCLA2>3.0.CO;2-F","article-title":"In situ hybridization: Methods and applications","volume":"11","author":"Jin","year":"1997","journal-title":"J. Clin. Lab. Anal."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"aaa6090","DOI":"10.1126\/science.aaa6090","article-title":"Spatially resolved, highly multiplexed RNA profiling in single cells","volume":"348","author":"Chen","year":"2015","journal-title":"Science"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1038\/s41586-019-1049-y","article-title":"Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH","volume":"568","author":"Eng","year":"2019","journal-title":"Nature"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1665","DOI":"10.1016\/j.cell.2020.10.026","article-title":"High-Spatial-Resolution Multi-Omics Sequencing via Deterministic Barcoding in Tissue","volume":"183","author":"Liu","year":"2020","journal-title":"Cell"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"100532","DOI":"10.1016\/j.xpro.2021.100532","article-title":"Spatial multi-omics sequencing for fixed tissue via DBiT-seq","volume":"2","author":"Su","year":"2021","journal-title":"STAR Protoc."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"5636","DOI":"10.1038\/s41467-021-25871-2","article-title":"High-throughput RNA sequencing of paraformaldehyde-fixed single cells","volume":"12","author":"Phan","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1038\/nmeth.3629","article-title":"Fixed single-cell transcriptomic characterization of human radial glial diversity","volume":"13","author":"Thomsen","year":"2016","journal-title":"Nat. Methods"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"100065","DOI":"10.1016\/j.xgen.2021.100065","article-title":"Genome-wide spatial expression profiling in formalin-fixed tissues","volume":"1","author":"Larsson","year":"2021","journal-title":"Cell Genom."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"177","DOI":"10.3109\/10520299409106282","article-title":"Kinetic Studies of Formaldehyde Binding in Tissue","volume":"69","author":"Helander","year":"1994","journal-title":"Biotech. Histochem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"482","DOI":"10.3389\/fonc.2019.00482","article-title":"Laser Capture Microdissection and RNA-Seq Analysis: High Sensitivity Approaches to Explain Histopathological Heterogeneity in Human Glioblastoma FFPE Archived Tissues","volume":"9","author":"Civita","year":"2019","journal-title":"Front. Oncol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1816","DOI":"10.1101\/gr.234807.118","article-title":"Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ","volume":"29","author":"Foley","year":"2019","journal-title":"Genome Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"687","DOI":"10.1016\/j.jmoldx.2011.06.007","article-title":"Paraffin embedding contributes to RNA aggregation, reduced RNA yield, and low RNA quality","volume":"13","author":"Evers","year":"2011","journal-title":"J. Mol. Diagn."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"von Ahlfen, S., Missel, A., Bendrat, K., and Schlumpberger, M. (2007). Determinants of RNA quality from FFPE samples. PLoS ONE, 2.","DOI":"10.1371\/journal.pone.0001261"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1016\/j.jhevol.2004.10.003","article-title":"Evolution of the brainstem orofacial motor system in primates: A comparative study of trigeminal, facial, and hypoglossal nuclei","volume":"48","author":"Sherwood","year":"2005","journal-title":"J. Hum. Evol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"222","DOI":"10.1002\/(SICI)1097-0185(19990201)254:2<222::AID-AR8>3.0.CO;2-B","article-title":"Organization of motoneurons in the dorsal hypoglossal nucleus that innervate the retrusor muscles of the tongue in the rat","volume":"254","author":"McClung","year":"1999","journal-title":"Anat. Rec."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/0197-4580(92)90003-G","article-title":"Progressive decline in spatial learning and integrity of forebrain cholinergic neurons in rats during aging","volume":"13","author":"Fischer","year":"1992","journal-title":"Neurobiol. Aging"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1038\/s41398-020-01087-8","article-title":"Ntrk1 mutation co-segregating with bipolar disorder and inherited kidney disease in a multiplex family causes defects in neuronal growth and depression-like behavior in mice","volume":"10","author":"Nakajima","year":"2020","journal-title":"Transl. Psychiatry"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1103","DOI":"10.1016\/j.ajhg.2012.09.019","article-title":"Defective presynaptic choline transport underlies hereditary motor neuropathy","volume":"91","author":"Barwick","year":"2012","journal-title":"Am. J. Hum. Genet."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1442","DOI":"10.1212\/WNL.0000000000003179","article-title":"Variants in SLC18A3, vesicular acetylcholine transporter, cause congenital myasthenic syndrome","volume":"87","author":"Verschuuren","year":"2016","journal-title":"Neurology"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1572","DOI":"10.1016\/j.celrep.2017.07.053","article-title":"NKX2-1 is required in the embryonic septum for cholinergic system development, learning, and memory","volume":"20","author":"Magno","year":"2017","journal-title":"Cell Rep."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1767","DOI":"10.1111\/j.1460-9568.2011.07890.x","article-title":"The integrity of cholinergic basal forebrain neurons depends on expression of Nkx2-1","volume":"34","author":"Magno","year":"2011","journal-title":"Eur. J. Neurosci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1016\/j.neuron.2009.06.026","article-title":"The Role of Apolipoprotein E in Alzheimer\u2019s Disease","volume":"63","author":"Kim","year":"2009","journal-title":"Neuron"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"164","DOI":"10.3389\/fnins.2019.00164","article-title":"Clusterin in Alzheimer\u2019s disease: Mechanisms, genetics, and lessons from other pathologies","volume":"13","author":"Foster","year":"2019","journal-title":"Front. Neurosci."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1016\/j.exger.2019.01.017","article-title":"p38 MAPK\u2013dependent alphaB-crystallin phosphorylation in Alzheimer\u2019s disease\u2013like pathology in OXYS rats","volume":"119","author":"Muraleva","year":"2019","journal-title":"Exp. Gerontol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1146\/annurev-med-042915-103753","article-title":"Update on Alzheimer\u2019s disease therapy and prevention strategies","volume":"68","author":"Graham","year":"2017","journal-title":"Annu. Rev. Med."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"777","DOI":"10.1126\/science.aag2590","article-title":"How neuroinflammation contributes to neurodegeneration","volume":"353","author":"Ransohoff","year":"2016","journal-title":"Science"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"12676","DOI":"10.1523\/JNEUROSCI.1243-10.2010","article-title":"Fibrillar Amyloid\u2014Activated Human Astroglia Kill Primary Human Neurons via Neutral Sphingomyelinase: Implications for Alzheimer\u2019s Disease","volume":"30","author":"Jana","year":"2010","journal-title":"J. Neurosci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/j.neuron.2014.11.018","article-title":"NF\u03baB-Activated Astroglial Release of Complement C3 Compromises Neuronal Morphology and Function Associated with Alzheimer\u2019s Disease","volume":"85","author":"Lian","year":"2015","journal-title":"Neuron"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"876","DOI":"10.1038\/s41593-022-01104-7","article-title":"A shared disease-associated oligodendrocyte signature among multiple CNS pathologies","volume":"25","author":"Kenigsbuch","year":"2022","journal-title":"Nat. Neurosci."},{"key":"ref_43","first-page":"532","article-title":"TRIzol: A new reagent for optimal single-step isolation of RNA","volume":"15","author":"Simms","year":"1993","journal-title":"Focus"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Amini, P., Ettlin, J., Opitz, L., Clementi, E., Malbon, A., and Markkanen, E. (2017). An optimised protocol for isolation of RNA from small sections of laser-capture microdissected FFPE tissue amenable for next-generation sequencing. BMC Mol. Biol., 18.","DOI":"10.1186\/s12867-017-0099-7"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1093\/toxsci\/kfx278","article-title":"Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples","volume":"162","author":"Wehmas","year":"2018","journal-title":"Toxicol. Sci."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"773","DOI":"10.1369\/jhc.5A6859.2006","article-title":"Improved RT-PCR Amplification for Molecular Analyses with Long-term Preserved Formalin-fixed, Paraffin-embedded Tissue Specimens","volume":"54","author":"Hamatani","year":"2006","journal-title":"J. Histochem. Cytochem."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"12335","DOI":"10.1038\/srep12335","article-title":"Robust gene expression and mutation analyses of RNA-sequencing of formalin-fixed diagnostic tumor samples","volume":"5","author":"Graw","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_48","first-page":"91","article-title":"Accurate RNA Sequencing From Formalin-Fixed Cancer Tissue to Represent High-Quality Transcriptome From Frozen Tissue","volume":"2","author":"Li","year":"2018","journal-title":"JCO Precis. Oncol."},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Li, P., Conley, A., Zhang, H., and Kim, H.L. (2014). Whole-Transcriptome profiling of formalin-fixed, paraffin-embedded renal cell carcinoma by RNA-seq. BMC Genom., 15.","DOI":"10.1186\/1471-2164-15-1087"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"202","DOI":"10.1093\/toxsci\/kfw161","article-title":"Editor\u2019s Highlight: Dose\u2013Response Analysis of RNA-Seq Profiles in Archival Formalin-Fixed Paraffin-Embedded Samples","volume":"154","author":"Hester","year":"2016","journal-title":"Toxicol. Sci."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1038\/nmeth.1294","article-title":"miRNA in situ hybridization in formaldehyde and EDC-fixed tissues","volume":"6","author":"Pena","year":"2009","journal-title":"Nat. Methods"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"792","DOI":"10.1016\/j.cell.2016.01.038","article-title":"Multiplexed Intact-Tissue Transcriptional Analysis at Cellular Resolution","volume":"164","author":"Sylwestrak","year":"2016","journal-title":"Cell"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"5141","DOI":"10.1007\/s12035-021-02453-3","article-title":"Profiling Basal Forebrain Cholinergic Neurons Reveals a Molecular Basis for Vulnerability Within the Ts65Dn Model of Down Syndrome and Alzheimer\u2019s Disease","volume":"58","author":"Alldred","year":"2021","journal-title":"Mol. Neurobiol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"3774","DOI":"10.1016\/j.neuron.2022.08.025","article-title":"Molecularly defined and functionally distinct cholinergic subnetworks","volume":"110","author":"Li","year":"2022","journal-title":"Neuron"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"566","DOI":"10.1038\/nprot.2017.003","article-title":"Spatial transcriptomic analysis of cryosectioned tissue samples with Geo-seq","volume":"12","author":"Chen","year":"2017","journal-title":"Nat. Protoc."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"12142","DOI":"10.1038\/ncomms12142","article-title":"High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellular level","volume":"7","author":"Gong","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"1067","DOI":"10.1002\/jemt.20836","article-title":"Low-temperature glycol methacrylate resin embedding method: A protocol suitable for bone marrow immunohistochemistry, PCR, and fish analysis","volume":"73","author":"Zhang","year":"2010","journal-title":"Microsc. Res. Tech."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"825","DOI":"10.1369\/jhc.2009.953695","article-title":"Localization of mRNAs and Proteins in Methyl Methacrylate-embedded Tissues","volume":"57","author":"Torgersen","year":"2009","journal-title":"J. Histochem. Cytochem."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"13891","DOI":"10.1038\/s41598-017-14360-6","article-title":"A platform for efficient identification of molecular phenotypes of brain-wide neural circuits","volume":"7","author":"Jiang","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"907","DOI":"10.1038\/s41587-019-0201-4","article-title":"Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype","volume":"37","author":"Kim","year":"2019","journal-title":"Nat. Biotechnol."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"giab008","DOI":"10.1093\/gigascience\/giab008","article-title":"Twelve years of SAMtools and BCFtools","volume":"10","author":"Danecek","year":"2021","journal-title":"GigaScience"},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"2078","DOI":"10.1093\/bioinformatics\/btp352","article-title":"The Sequence Alignment\/Map format and SAMtools","volume":"25","author":"Li","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"2184","DOI":"10.1093\/bioinformatics\/bts356","article-title":"RSeQC: Quality control of RNA-seq experiments","volume":"28","author":"Wang","year":"2012","journal-title":"Bioinformatics"},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1038\/nbt.1754","article-title":"Integrative genomics viewer","volume":"29","author":"Robinson","year":"2011","journal-title":"Nat. Biotechnol."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"923","DOI":"10.1093\/bioinformatics\/btt656","article-title":"featureCounts: An efficient general purpose program for assigning sequence reads to genomic features","volume":"30","author":"Liao","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"511","DOI":"10.1038\/nbt.1621","article-title":"Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation","volume":"28","author":"Trapnell","year":"2010","journal-title":"Nat. Biotechnol."},{"key":"ref_67","doi-asserted-by":"crossref","unstructured":"Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol., 15.","DOI":"10.1186\/s13059-014-0550-8"},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"284","DOI":"10.1089\/omi.2011.0118","article-title":"clusterProfiler: An R package for comparing biological themes among gene clusters","volume":"16","author":"Yu","year":"2012","journal-title":"OMICS"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/12\/6504\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:57:46Z","timestamp":1760108266000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/12\/6504"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,6,13]]},"references-count":68,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2024,6]]}},"alternative-id":["ijms25126504"],"URL":"https:\/\/doi.org\/10.3390\/ijms25126504","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,6,13]]}}}